Methods of constructing camel antibody libraries

ABSTRACT

The present invention provides camel antibody libraries that maintain in vivo diversity of camelid antibody variable region genes. The in vivo diversity of antibody variable region genes can be accomplished by, for example, mixing genes derived from a plurality of animals or modifying gene amplification conditions. Conventional methods yield only VHHs with limited repertoire diversity. However, the present invention provides libraries comprising genes encoding functional VHHs with sufficient repertoire size. According to the present invention, libraries that enable to freely obtain VHHs against arbitrary antigens are provided. VHHs have excellent solubility and stability, and show a reactivity that usually cannot be expected from tetrameric IgGs.

TECHNICAL FIELD

The present invention relates to methods of constructing camel antibodylibraries.

BACKGROUND ART

Two structures of IgGs constituting the immunoglobulins (antibodymolecules) of camelids are known to exist: one a heterotetramer havingheavy chains and light chains, and the other consisting of a heavy-chaindimer (Isr. J. Vet. Med. 43(3), 198 (1987); Hamers-Casterman et al.,Nature, 363, 446 (1993)). The tetrameric structure is a commoncharacteristic of IgGs among humans and most animals. On the other hand,the latter IgG having a heavy-chain dimer structure is consideredcharacteristic of camelids. The IgGs consisting of a heavy chain dimerof camelids does not accidentally result from pathologic conditions.

Immunoglobulins lacking light chains have been found in Camelusbactrianus and Camelus dromedarius, which are Asian and Africancamelids, as well as in all species of South American camelids. SouthAmerican camelids include Lama pacos, Lama glama, and Lama vicugna. Themolecular weight of dimeric IgG differs depending on the animal species.The molecular weight of heavy chains constituting these immunoglobulinsis approximately 43 kDa to approximately 47 kDa, and normally are 45kDa.

Another characteristic of the heavy-chain dimer IgG is that thisantibody lacks the first domain of the constant region called CH1according to the definition by Roitt et al. Furthermore, the hingeregion has an amino acid sequence different from that of a normalheterotetrameric antibody (heavy chains+light chains). Based on thedifferences in the amino acid sequences, the IgGs of dromedaries areclassified as follows (Hamers-Casterman et al., Nature 363, 446 (1993)):

-   IgG2: comprising a long hinge sequence (SEQ ID NO: 8);-   IgG3: comprising a short hinge sequence (SEQ ID NO: 9); and-   IgG1: heterotetrameric antibody.

Since the VH region of a heavy chain dimer IgG does not have to makehydrophobic interactions with a light chain, the region in the heavychain that normally contacts a light chain is mutated to hydrophilicamino acid residues. Due to structural differences compared to VHs ofnormal heterotetrameric IgGs, VH domains of the heavy-chain dimer IgGsare called Variable domain of the heavy-chain of heavy-chain antibody(VHH).

VHH has excellent solubility due to its hydrophilic amino acid residues.Amino acid substitutions are scattered throughout the primary structure(amino acid sequence) of VHH. Additionally, these hydrophilic amino acidresidues form a cluster in the space of the tertiary structure of VHcorresponding to the site that interacts with the VL domain. Herein, theaforementioned space of the tertiary structure is specifically calledformer VL side. These amino acid substitutions are, for example, V37F orV37Y, G44E, L45R or L45C, and W47 are also mostly substituted with Gly.Such substitutions increase the hydrophilicity of the former VL. side ofVHH.

Therefore, the solubility of VHH is much higher than that of VH isolatedand purified from humans or mice (single domain antibody; Ward et al.,Nature, 341, 544 (1989)). VHH can be easily concentrated to 10 mg/mL inordinary buffer solutions without any signs of aggregation. Thisconcentration corresponds to, for example, approximately 100 times thesolubility of mouse VH.

Furthermore, VHHs derived from camels and llamas have very highthermostability compared to mouse heterotetrameric antibodies. The useof VHH derived from these species can provide, for example, moleculesthat maintain their antigen binding ability even at 90° C. (van derLinden et al., Biochim. Biophys. Acta 1431 (1), 37 (1999))

The diversity of antibody repertoire of camelids is determined by thecomplementary determining regions (CDR) 1, 2, and 3 in the VH or VHHregions. Possession of three CDRs is in common with the IgGs of otheranimal species. However, the CDR3 in the camel VHH region ischaracterized by its relatively long length averaging 16 amino acids(Muyldermans et al., Protein Engineering 7(9), 1129 (1994)). Forexample, compared to the CDR3 of mouse VH having an average of 9 aminoacids, the CDR3 of camel IgG is very long.

Most antigen binding sites of structurally known heavy chains+lightchains heterotetrameric antibodies are known to form antigen-bindingsurfaces such as grooves, cavities, and flat areas (Webster et al.,Current Opinion in Structural Biology 4, 23 (1994)). Therefore, when anepitope of a substance to be bound also forms a groove or a cavity, theantigen-binding site of the antibody may not bind well. For example, inproteins including enzymes, catalytic or functional residue, or toxicregion is often located at the interior of a cleft. This structurefacilitates extremely specific interactions of enzyme substrates andreceptors with proteins. However, structures such as cavities and cleftsare difficult for heterotetrameric antibodies to recognize, andtherefore, they do not have high immunogenicity.

In contrast, there are reports that VHHs of camelids can specificallyrecognize clefts and cavities due to their characteristic structuredescribed above. For example, in an experiment where antibodies wereisolated from peripheral blood of camels immunized with an enzyme as theantigen, antibodies that seal the enzyme active center existed onlyamong the camel IgG2 and IgG3, and not in IgG1 (Lauwereys et al., EMBOJ. 17(13), 3512 (1998)). Furthermore, VHH having lysozyme activityinhibitory effect was isolated by the phage display method from alibrary derived from camels immunized with lysozyme (Arbabi Ghahroudi etal., FEBS Letters 414, 521 (1997)). The structure of the isolated VHH incomplex with lysozyme was elucidated by X-ray crystallographic analysis(Desmyter et al., Nature Structural Biology 2, 803 (1996)). The resultsshowed that in IgG2 or IgG3 of the camel antibody, the CDR3 regionhaving a long protrusion is inserted and bound to the substrate-bindingsite of the enzyme such that the active center is sealed to causecompetitive inhibition.

Industrially useful characteristics can be found in VHHs derived fromcamelids such as high solubility and the possible existence of novelactivity that cannot be expected from tetrameric IgGs. To obtain anantibody variable region, an animal must be immunized with an antigen ofinterest to separate an antibody. However, such a classical methodinvolves problems such as the need to purify large amounts of antigensand generation of non-specific antibodies. Accordingly, as a method formore easily obtaining an antibody variable region, a screening methodusing an rgdp library has been proposed. The phrase “rgdp library”refers to a library consisting of a genetic display package whereingenes encoding substances with binding affinity, such as antibodyvariable regions, display their expression products. A representativeexample of an rgdp library includes a phage library that displaysantibody variable regions.

The method of obtaining antibodies using a phage library displayingantibody variable regions is being noticed as a novel method forobtaining antibodies that succeed to labor-intensive, classical methodsof antibody production. The present inventors have also constructednovel antibody libraries that allow efficient acquisition of antibodyvariable regions, and have already filed a patent application (WO01/62907). It would also be useful for VHHs to construct a library thatallows to freely select a VHH with a binding affinity towards anarbitrary antigen from the library. However, several problems have beenpointed out in the construction of camelid VHH libraries.

Another characteristic of the structure of camelid VHH is that it oftencontains a cysteine residue in the CDR3 in addition to cysteinesnormally existing at positions 22 and 92 of the variable region. Thecysteine residues in CDR3 are considered to form disulfide bonds withother cysteines in the vicinity of CDR1 or CDR2 (Muyldermans et al.,Protein Engineering 7(9), 1129 (1994); Muyldermans et al., J. Mol.Recognit. 12, 131 (1999)). CDR1 and CDR2 are determined by the germlineV gene. They play important roles together with CDR3 in antigen binding(Desmyter et al., Nature Structural Biology, 2, 803 (1996); Structure7(4), (1999); Spinelli et al., J. Mol. Biol. 311(1), 123 (2001)). Ingeneral, the term “germline” refers to chromosomal genes maintained ingerm cells, i.e., chromosomal genes that have not undergonerearrangement. Herein, among the chromosomal genes, particularly theregion that constitutes the antibody gene is referred to as germline.

Recently, germlines of dromedaries and llamas belonging to Camelidiaewere studied. As a result, IgGs of dromedaries and llamas wereclassified according to the length of CDR2 and cysteine positions in theV region (Nguyen et al., EMBO J. 19(5), 921 (2000); Harmsen et al., Mol.Immunol. 37, 579 (2000)).

However, it has been noted that the antibody genes of the entiregermline of a dromedary cannot be considered as sufficiently covered byhitherto obtained antibody genes. For example, the concentration of theclassification of cDNA nucleotide sequences of the antibodies obtainedso far on particular classes reveal that the germlines from which theseantibodies were derived had been biased (Nguyen et al., EMBO J. 19 (5),921 (2000)). Furthermore, methodologically, they were considered toimply problems as follows. Specifically, known libraries wereconstructed using only one type of primer as the N-terminal primer.Therefore, due to problems of specificity, some germlines from which theVHH genes are derived might have leaked, or the amplification productsmight have been biased (Arbabi Ghahroudi et al., FEBS Letters 414, 521(1997)).

A library having biased constituent genes is poor in repertoire.Therefore, screening of such a library may not yield antibodies againstan antigen of interest. This may be the reason why antibodies inhibitingor promoting an enzyme activity could not be obtained from phagelibraries derived from non-immunized camels.

Prior art proposes methods to immunize camels or llamas in advance witha sufficient amount of antigen in order to obtain the variable region ofimmunoglobulin heavy chains of camels or llamas (Published JapaneseTranslation of International Publication No. Hei 11-503918; Lauwereys etal., EMBO J. 17(13), 3512 (1998); Arbabi Ghahroudi et al., FEBS Letters414, 521 (1997)). This method utilizes the phenomenon that the immunesystems of camels and llamas mature their own heavy chain antibodies invivo (Published Japanese Translation of International Publication No.2000-515002; J. Immuno. Methods 240, 185 (2000)). Based on this method,antibodies that recognize lysozyme, tetanus toxoid, carbonic anhydrase,amylase, RNaseA, azo dye and such have been obtained.

However, the need of immunological sensitization in this method imposesvarious restrictions such as those described below:

necessity of immunological sensitization period;

toxic influence of immunogens on camelids;

difficulty in obtaining antibodies against substances with lowimmunogenicity; and

necessity of relatively large amounts of antigens for immunologicalsensitization.

Furthermore, to avoid the problems of immunological sensitization, amethod comprising the following steps was proposed (Published JapaneseTranslation of International Publication No. 2000-515002):

-   1) randomly selecting camelid heavy-chain antibodies;-   2) isolating coding sequences and cloning them into phage display    vectors;-   3) modifying those coding sequences in at least one codon by random    substitution;-   4) constructing a library of the randomly mutated coding sequences    in the phage display vectors;-   5) expressing the coding sequences in phages transfected with those    vectors; and-   6) subsequently, sorting the phages with immobilized antigen to    select recognition molecules specific to the antigen.

As an alternate solution, a method using the framework of camel antibodyhas also been suggested. According to this method, camel antibodies arereconstructed by incorporating CDR1, CDR2, and CDR3 of VHH and VH intothe camel antibody framework. This method applies the method developedfor humanizing mouse VH. The loops of each CDR can be mutated randomlyto enlarge the repertoire size. As a result, the affinity andspecificity of the antibodies can be controlled (Published JapaneseTranslation of International Publication No. 2000-515002).

All of these solutions are based on the principle of aiming to attaindiversity by introducing artificial mutations into the coding sequences.However, most of these attempts require a great deal of effort andcomplicated procedures to introduce the mutations, and takes a longtime. Furthermore, many of such attempts accompanied inefficiency ofproducing overhigh inactive antibodies along with the production ofactive antibodies.

A conceivable alternative method involves constructing a phage libraryby incorporating VHH genes obtained from tissues and blood ofnon-immunized camels into phage display vectors, and selectingrecognition molecules specific to an antigen by selecting phages thathave binding ability from the phage library using the immobilizedantigen. However, this method had been contemplated to only yield VHHsagainst substances with sufficient immunogenicity. Therefore, methodsusing phage libraries incorporating VHH genes had not been sufficientlystudied (Published Japanese Translation of International Publication No.2000-515002). Accordingly, non-immunized camel-derived VHH antibodyphage libraries comprising a repertoire diverse enough to yieldantibodies inhibiting or promoting enzyme activities had not existed.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide VHH libraries havinga sufficient repertoire size. Furthermore, another objective of thepresent invention is to provide VHH libraries that do not accompanyproduction of inactive VHH.

The present invention provides VHH libraries having a very largediversified repertoire by improving the conventional process ofisolating VHH variable regions. More specifically, by devising primersfor amplifying genes, germline VHH genes could be amplified that muchfaithfully reproduce the in vivo diversity.

Furthermore, the present inventors showed that VHHs are antibodymolecules developed in camelids having mainly supplementary importance,and are a group whose antibody maturation process completely differsfrom that of VH genes. Therefore, to maximize the diversity of VHHgenes, the present inventors aimed to ensure diversity by increasing thenumber of animals, which had not been emphasized in previous libraries.As a result, a library with rich diversity was successfully constructedusing camel genes from more than one animal.

Moreover, to minimize the loss of diversity during amplification ofgenes by PCR, the present inventors collected the genes amplified by PCRin the exponential phase, and avoided saturation of some of the genes.In addition, the present inventors also obtained the variable regiongenes of the IgM class. Generally, it is suggested that naiverepertoires are included in the immunoglobulins of the IgM class. IgGsmay accompany bias of clones due to natural immunological sensitizationof camels in vivo. Meanwhile, such bias is considered not to occur inIgM.

According to such processes, non-immunized antibody variable regionlibraries that ensure extremely large diversity can be constructed.Thus, the present invention relates to antibody variable regionlibraries, methods for constructing them, and uses thereof as follows:

[1] a library of camelid-derived VHHs, which maintains the in vivodiversity of variable regions in a camelid;

[2] the library of [1], wherein 33 arbitrary clones selected from clonesconstituting the library comprise genes belonging to at least 8 or moreclasses;

[3] the library of [2], wherein a sufficient amount of clones randomlyselected from clones constituting the library comprise genes of at least6 VHH subfamilies, and at the same time genes belonging to 15 or moreclasses;

[4] the library of [1] comprising at least 10⁵ or more VHH gene clones;

[5] the library of [1] consisting of VHH gene clones derived fromimmunoglobulin genes of IgG2 and/or IgG3;

[6] the library of [5], wherein the VHH gene rate in the library is 60%or more;

[7] the library of [1], which is an rgdp library, [8] a method ofobtaining a gene encoding a VHH that has an affinity for a substance ofinterest, which comprises the steps of:

(1) contacting the library of [7) with the substance of interest, and

(2) selecting a clone encoding a VHH that binds to the substance ofinterest;

[9] the method of [8], wherein the substance of interest is an enzymemolecule or a fragment thereof;

[10] a method of obtaining a VHH that has a function to regulate anenzyme activity, which comprises the steps of:

(1) obtaining a VHH that binds to an enzyme by the method of [9],

(2) contacting the VHH obtained in step (1) with the enzyme, and

(3) selecting the VHH that has a function to modify the enzyme activityof said enzyme compared to that in the absence of the VHH;

[11] a gene encoding the VHH selected by the method of (8] or [10];

[12] a method of producing an immunoglobulin comprising acamelid-derived VHH as a variable region, or a fragment thereof, whichcomprises the steps of:

(1) obtaining a gene encoding a VHH that has an binding activity for asubstance of interest by the method of [8],

(2) preparing a VHH expression vector by incorporating the obtainedVHH-encoding gene into a vector expressible in a host cell, and

(3) introducing the VHH expression vector into the host cell to collectproteins comprising the VHH from the culture;

[13] a method of constructing a VHH library, which comprises the stepsof:

(1) obtaining VHH genes from a plurality of animals belonging toCamelidae, and

(2) preparing a library by mixing the VHH genes obtained in step (1);

[14] the method of [13], which comprises the step of amplifying the VHHgenes obtained in step (1);

[15] the method of [14), wherein the amplification is-performed by PCR;

[16] the method of [15], which comprises the step of collectingamplification products of the PCR during the exponential phase;

(17] the method of [15], wherein the animals of Camelidae aredromedaries and the PCR is performed using primer sets consisting of a5′ primer selected from any one of the oligonucleotides having thenucleotide sequences of SEQ ID NOs: 1 to 6, and a 3′ primer consistingof an oligonucleotide having the nucleotide sequence of SEQ ID NO: 10,and which comprises the step of mixing the amplification products fromeach of the primer sets;

[18] the method of [15], wherein the animals of Camelidae aredromedaries, and the PCR is performed using primer sets consisting of a5′ primer selected from any one of the oligonucleotides having thenucleotide sequences of SEQ ID NOs: 1 to 6, and a 3′ primer consistingof an oligonucleotide having the nucleotide sequence of SEQ ID NO: 11,and which comprises the step of mixing amplification products from eachof the primer sets;

[19] the method of [17] or [18] comprising the step of digesting theamplification products with restriction enzymes SfiI and AscI, andligating the digested products into vectors having features (i) and (ii)as follows:

(i) comprising a SfiI site and an AscI site; and

(ii) upon transformation of the vector into an appropriate host,expressing a protein encoded by an exogenous gene inserted into the siteof (i) as a fusion protein with a protein constituting a phage;

[20] a VHH library, which can be constructed by the method of [17] or[18];

[21] a primer set for camel VHH gene amplification consisting of a 5′primer selected from oligonucleotides having the nucleotide sequences ofSEQ ID NOs: 1 to 6, and a 3′ primer selected from oligonucleotideshaving the nucleotide sequences of SEQ ID NOs: 10 and 11, respectively;

[22] a method of constructing a VH library, which comprises the stepsof:

(1) obtaining VH genes from a plurality of animals of Camelidae, and

(2) preparing a library by mixing the VH genes obtained in step (1);

[23) the method of [22] comprising the step of amplifying the VH genesobtained in step (1);

[24] the method of [23], wherein the amplification is performed by PCR;

[25] the method of [24] comprising the step of collecting theamplification products of the PCR during the exponential phase;

[26] the method of [24], wherein the animals of Camelidae aredromedaries, and the PCR is performed using primer sets consisting of a5′ primer selected from any one of the oligonucleotides having thenucleotide sequences of SEQ ID NOs: 1 to 6 and a 3′ primer consisting ofan oligonucleotide having the nucleotide sequence of SEQ ID NO: 41, andwhich comprises the step of mixing the amplification products from eachof the primer sets;

[27] the method of [26] comprising the step of digesting theamplification products with restriction enzymes SfiI and AscI andligating the digested products to a vector having features (i) and (ii)as follows:

(i) comprising a SfiI site and an AscI site, and

(ii) upon transformation of the vector into an appropriate host,expressing a protein encoded by an exogenous gene inserted into the siteof (i) as a fusion protein with a protein constituting a phage;

[28] a VH library derived from camelid IgM;

[29] a VH library obtainable by the method of [22];

(30] the library of [28] or [29], which is an rgdp library;

[31] a method of obtaining a gene encoding a VH that has an affinity fora substance of interest, which comprises the steps of:

(1) contacting the library of [30] with the substance of interest, and

(2) selecting a clone comprising a VH that binds to the substance ofinterest;

[32] the method of [31], wherein the substance of interest is an enzymemolecule or a fragment thereof;

[33] a method of obtaining a VH comprising a function to regulate anenzyme activity, which comprises the steps of:

(1) obtaining a VH that binds to an enzyme by the method of [31],

(2) contacting the VH obtained in step (1) with the enzyme, and

(3) selecting the VH that has a function to modify the enzyme activityof the enzyme compared to that in the absence of the VH;

[34] a gene encoding a VH selected by the method of [31];

[35] a method of producing an immunoglobulin comprising adromedary-derived VH as a variable region, or a fragment thereof, whichcomprises the steps of:

(1) obtaining a gene encoding a VH having a binding activity for asubstance of interest by the method of [31],

(2) preparing a VH expression vector by incorporating the obtainedVH-encoding gene into a vector expressible in a host cell, and

(3) introducing the VH expression vector into the host cell to collectproteins comprising the VH from the culture; and

[36] a primer set for dromedary VH gene amplification that consists of a5′ primer selected from any one of the oligonucleotides having thenucleotide sequences of SEQ ID NOs:1 to 6, and a 3′ primer consisting ofan oligonucleotide having the nucleotide sequence of SEQ ID NO: 41.

The present invention relates to libraries of camelid-derived antibodyvariable regions, which maintain the in vivo diversity of the variableregions of a camelid. A VHH library comprising VHH genes as variableregion genes can be confirmed to maintain the in vivo diversity asfollows. Specifically, 33 arbitrary clones may be selected from clonesconstituting a library, and when the selected clones are determined tocomprise genes belonging to at least 8 or more classes, this library canbe assumed to maintain the in vivo diversity. More specifically,sufficient amount of clones may be randomly selected from clonesconstituting a library, and when the selected clones are determined tocomprise genes of preferably 6 VHH subfamilies and at the same timegenes belonging to 15 or more classes, this library can be assumed tomaintain the in vivo VHH diversity.

In the present invention, the VHH subfamilies are classified based onthe positions of cysteine residues. On the other hand, the classes aregrouped based on the position of cysteine residues, the length of CDR2,and the length of CDR1. In Table 1 shown in Example 2, each framecorresponds to one class.

The conventional subfamily classification of VHHs (Nguyen et al., EMBOJ. 19(5), 921 (2000); Mol. Immunol. 127(10), (2000)) is based on thepositions of cysteine residues and the length of CDR2. However,according to the analysis by the present inventors, cysteine residuesand CDR2 lengths that could not be covered by the conventionalclassification were found. Furthermore, though the number of amino acidresidues constituting CDR1 had been considered to be restricted to onevalue, CDR1 with different numbers were found according to the analysisof the present inventors. Therefore, VHHs cannot be virtually classifiedbased on the conventional classification.

Applying the method of classification of subfamilies and classes of thisinvention to a known library analysis result (Nguyen et al., EMBO J.19(5), 921 (2000)), 5 subfamilies and 7 classes (from 72 clones) werefound. On the other hand, following results were obtained for the VHHlibraries of the present invention, for example, when the classificationmethod of this invention was applied to the analysis result of clonesconstituting VHH libraries constructed in the Examples described later.Results of analyzing 89 arbitrary clones from IgG2-derived VHH library:

number of subfamilies: 7 number of classes: 31 Results of analyzing 59arbitrary clones from IgG3-derived VHH library:

number of subfamilies: 7 number of classes: 20

Based on these findings, the present inventors deemed that a librarycould be judged to maintain a sufficient diversity when 50 or morearbitrary clones constituting the library are determined to containgenes of 6 or more subfamilies and 15 or more classes, based on theclassification of the present invention.

Specifically, a VHH library of the present invention preferablycomprises, for example, 10⁵ or more clones. Therefore, a non-immunizedcamel antibody library preferably comprises at least 10⁵ or more clones.More preferably, the antibody libraries of the present invention can beprepared as practical libraries by comprising 10⁶ or more, normally 10⁷or more, or 10⁸ or more, even more preferably 10⁹ or more, and ideally10¹⁰ or more clones.

For example, a camel antibody library of the present inventionobtainable by methods such as those mentioned later comprises 10¹⁰ ormore clones. A VHH gene library with such a rich variety has not beenreported so far. More importantly, the percentage of normal genes in theVHH genes constituting the libraries of this invention is extremelyhigh. Whether the VHH genes are normal or not can be confirmed bydetermining their nucleotide sequence and by analyzing followingqualities:

presence of a framework with a nucleotide sequence highly homologous toknown camel antibody framework;

absence of frame shift in the amino acid sequence to be translated; and

no generation of a stop codon.

More preferably, it is also an important factor for normal VHH genes toencode a protein having hydrophilic amino acid residues at the positionof the hinge region where the VHH-characteristic hydrophilic amino acidsare located.

Preferably, 80% or more, more preferably 85% or more, even morepreferably 90% or more, and particularly preferably 95% or more of theVHH genes constituting the libraries of the present invention arenormal. More specifically, the present invention relates to VHHlibraries comprising 90% or more normal IgG2-derived VHH genes.Alternatively, the present invention relates to VHH libraries comprising95% or more normal IgG3-derived VHH genes. A library with theaforementioned high diversity and comprising normal VHH genes in suchhigh proportions cannot be obtained according to conventional methods.

In the present invention, “VHH” refers to the variable regionconstituting an immunoglobulin having a dimeric structure that is foundin the blood of camelids. VHH genes constituting the libraries of thepresent invention may comprise a constant region in addition to thevariable region. Therefore, the VHH genes may accompany the nucleotidesequence of a hinge region. Since VHHs and VHs often show structuraldifferences in the hinge regions, VHH genes can be specificallycollected by setting primers at the hinge region in order to obtain VHHgenes carrying the hinge region.

Accordingly, in the Examples described below, 3′ (C-terminal) primerswere set in the hinge region. As a result, VHH of the desired class waspresent at a high percentage as shown below in each group of VHH genesamplified by primers used in the Examples.

IgG2 (SEQ ID NO: 10) VH:VHH=7:91 (93% VHH)

IgG3 (SEQ ID NO: 11) VH:VHH=1:167 (99% VHH)

IgM (SEQ ID NO: 41) VH:VHH=189:3 (1.6% VHH)

The VHH libraries of the present invention can be constructed usingtechniques, for example, as below. These techniques are all useful forsupplementing the lack in repertoire size of VHHs.

(1) Utilizing VHH genes derived from a plurality of individuals.

(2) Amplifying VHH genes using primers that allow amplification of awide variety of genes.

(3) In the interest of amplifying VHH genes, collecting amplificationproducts during the progress of exponential amplification.

In the following, these techniques will be described more specifically.

-   (1) Utilizing VHH genes derived from a plurality of individuals

According to the findings of the present inventors, the repertoire sizeof camelid VHH genes is often limited and biased. Therefore,construction of a library with a repertoire size allowing optionalacquisition of an antibody against an arbitrary antigen is difficultusing VHH genes from one individual alone. Thus, the use of VHH genesfrom more than one individual to construct a library allows effectiveenlargement of the repertoire size.

The phrase “VHH genes of more than one individual” indicates that itcomprises VHH genes obtained from a plurality of genetically differentindividuals. The phrase “a plurality of genetically differentindividuals” refers to individuals with genetic differences at thegenomic level. Therefore, even if the individuals are littermates, theyare genetically different individuals when they are not monozygotic.However, for the purpose of increasing the repertoire size of a library,it is advantageous to combine individuals with more distant geneticrelationships.

In the present invention, VHH genes of more than one individual can beobtained by adding VHH genes from one individual to VHH genes derivedfrom another individual. There are no limitations on the VHH genes to beadded. Therefore, for example, even when a partial group of VHH genesderived from another individual is added, such collection of VHH genesare also encompassed by the present invention.

VHH genes to be added are preferably collected from 10⁵ or more cellsper individual, and it is desirable to add the entire group of VHH genesobtained from each individual. By utilizing the entire group of genes,bias of genes can be prevented. Since the bias of genes causes decreasein the repertoire size, it is effective to add, if possible, the entiregroup of VHH genes. The phrase “entire group of VHH genes” refers to allof the obtained VHH genes. Specifically, the present inventionencompasses libraries wherein not only all VHH genes from a certainindividual are added, but also those wherein all obtainable VHH genesare added.

Furthermore, in the present invention, VHH genes derived from anotherindividual that are added to VHH genes derived from a certain individualare preferably added evenly to avoid bias between the individuals. Thepurpose of mixing the VHH genes is to enlarge the repertoire size. Whenthe VHH genes are biased between individuals in a library, thepossibility increases that the VHH genes derived from a particularindividual are preferentially selected in the screening using thelibrary. Under such a circumstance, the effect of enlarging therepertoire size will be diminished. Therefore, it is important todecrease the bias between individuals. Thus, when adding VHH genes fromanother individual to VHH genes derived from a certain individual, theyare preferably added at an equal amount to the VHH genes derived fromthe certain individual. Furthermore, when mixing VHH genes derived froma plurality of individuals, it is preferred to use equal amounts of VHHgenes from each individual.

-   (2) Amplifying VHH genes using primers that allow amplification of    wider variety of genes

When an amount of mRNAs required for constructing a library can beobtained from an individual, a library can be constructed as it is.However, since the amount of mRNAs obtainable from an individual isnormally very small, the mRNAs are amplified to construct a VHH genelibrary. At this time, unless every single VHH gene is amplified, therepertoire size of the library cannot be enlarged.

In the present invention, there are no limitations on the methods ofgene amplification. Any method may be used as long as it allowsamplification of the group of VHH genes as thoroughly as possible. Whenusing PCR as the method of gene amplification, primers expected toamplify a wide variety of VHH genes are used. Primers for obtaining VHHgenes of camels and llamas are well known in the art. However, accordingto the knowledge of the present inventors, amplification of VHH geneswithout bias is difficult when known primers are used alone.

Therefore, the present inventors newly designed primers that allowamplification of VHH genes with less bias. The nucleotide sequences ofN-terminal (5′-side) primers for amplifying dromedary VHH genes designedby the present inventors are described in SEQ ID NOs: 1 to 6.Furthermore, primers that can anneald between the hinge region and CH3can be used as C-terminal (3′-side) primers. Such primers are well knownin the art. For example, the present inventors analyzed the amino acidsequences of the hinge regions of IgG2 and IgG3, and designed primersconsisting of the nucleotide sequences of SEQ ID NOs: 10 and 11, forIgG2 and IgG3, respectively. These primers can be used in 12 differentcombinations as primer sets, where each of the 6 kinds of 5′ primers iscombined with either of the 2 kinds of 3′ primers.

Apart from dromedary, to amplify VHH genes of Camelidae, the nucleotidesequences of VHH genes of interest may be analyzed by the analysismethods described in the Examples to design primers.

Some antibodies with VH structures that regulate the activity of anenzyme have been obtained. Therefore, the present inventors featured onthe antigen recognition repertoire of IgM. IgM can be considered to be aprototype of the variable region at the stage without antigenicstimulation, when complementing the antigen recognition repertoire ofVHHs of IgG with the variable regions of IgM. During the in vivomaturation of heavy chains upon immunization, IgM retains the prototypesof variable regions against all kinds of antigens. Therefore, thepresent inventors considered that IgM maintains the blueprint forproducing IgG variable regions optimized for an antigen by class change.Accordingly, the present inventors newly utilized a camel μ-chainrecognition sequence (SEQ ID NO: 41) to successfully isolate thevariable region of IgM. This IgM-derived heavy chain variable regionlibrary can further enlarge the diversity of the repertoire and isuseful for isolating recognition molecules and enzyme activityregulating molecules. In the present invention, the use of mRNAs derivedfrom a plurality of individuals is also effective in constructing alibrary consisting of IgM-derived VHs to maintain a highly diverserepertoire.

IgM-derived VH libraries have a complementary meaning to the VHHrepertoire derived from IgG2 or IgG3. “Complementary” means that theysupplement antibody variable regions having functions that cannot beselected from VHHs or are difficult to select due to small populationamong VHHs. Antibody variable regions that can recognize a wider varietyof molecules can be obtained by combining these libraries. Morespecifically, antibody variable regions having functions to regulateenzyme activities of a greater variety of enzymes can be isolated.

Combining these primers and using mRNAs obtained from camelantibody-producing cells as templates, VHH genes (mainly comprising VHgenes for IgM) are amplified by PCR. Splenocytes, peripheral blood Bcells, and such can be used as the antibody-producing cells. Theamplified VHH genes are collected to construct a VHH library of thepresent invention.

In the present invention, a large repertoire size is achieved by addingVHH genes of another individual to VHH genes derived from a certainindividual. For this purpose, amplification products may be mixed, oralternatively, pre-mixed mRNAs derived from a plurality of individualsmay be used as templates to amplify the genes. Collecting a fixed amountof mRNAs from a plurality of individuals and amplifying genes usingmixtures of the mRNAs as templates, the bias of genes among individualscan be diminished. Furthermore, this method is reasonable since theoperations of amplifying and collecting genes only need to be performedonce.

More specifically, mRNAs are collected from a plurality of camels andequal amounts of the mRNAs are mixed to produce an mRNA pool. cDNAs aresynthesized from this mRNApool using random primers, oligo-dT primers,primers. homologous to a constant region, or such, and used as templatesfor PCR. In the PCR, as described in (3), amplification products arecollected at the exponential phase.

The VHH libraries of the present invention can be separated into IgG2and IgG3 libraries, or each of the libraries can be mixed to form asingle library. Furthermore, when combining VH libraries of IgM, each ofthe libraries can be prepared as separate libraries, or each of thelibraries can be mixed to form a single library. When forming thelibraries separately, libraries of IgG2, IgG3, and IgM can be preparedby mixing amplification products of the same 3′ primers among theaforementioned primers.

Constructing a library for each of the respective classes of VHHsderived from IgG2 or IgG3, or VHs derived from IgM allows screening ofVHHs (or VHs) unaffected by other classes. For example, the presentinventors found that when IgG2 and IgG3 were mixed for the screening,IgG2 tended to be preferentially selected in certain cases. This mayoccur due to the differences in the expression level of each of theclasses, the differences in the number of constituent clones, and such.,Separating the libraries by class increases the possibility to obtainunbiased antibodies from each of the classes that has the function ofinterest without such interference of VHHs between classes.

Alternatively, when amplified VHH gene products are mixed afteramplification from each of the individuals, the bias of genes isprevented by maintaining a constant ratio of genes among individuals.More specifically, conditions for gene amplification such as the amountof templates, the combination of primers, and the number of PCRreactions are strictly matched among the individuals. Furthermore, byevenly mixing the amplification products, the bias of genes can beprevented. The absence of bias in a library signifies that the in vivogermline ratio of VHH gene expression products is maintained in thelibrary.

VHH genes are formed by the rearrangement of VH-D-JH genes in thegermline. Theoretically, by investigating clones constituting a VHH geneexpression product, it is possible to determine from which gene segmentwithin the germline the VH, D, and JH of the VHH gene are derived.Furthermore, the investigation of a plurality of clones allows toestimate the in vivo frequency of each gene segment with respect to allof the expression products. Herein, thus determined frequency estimatedfor each gene segment in all of the expression products is referred toas “germline ratio”.

In practice, since the genetic sequence of each of the gene segmentshave not been completely elucidated, germline ratios were analyzed basedon the classifications of “subfamily” or “class” described later. Eachof the gene segments can be classified into a “subfamily” or a “class”according to characteristics such as the length of CDR sequences, andCys positions. In a biased library, the expression frequency of clonesderived from a certain gene segment may be different from that in vivo.Therefore, the germline ratio can be used for evaluating libraries.

Twelve primer sets used by the present inventors can amplify anygermline gene that may constitute a VHH gene. Specifically, using theabove-mentioned primers, construction of libraries maintaining the invivo ratio of germlines from which VHHs are derived is possible.

The diversity of VHH is considered to come from the combination of threegene segments, VH-D-JH, constituting the antibody gene, addition anddeletion of nucleotides during the rearrangement process of these genesegments, and mutations in antibody producing cells. These mechanismsare believed to be the same as the general mechanism for obtainingantibody diversity in mammals.

Among the mechanisms supporting the diversity of VHHs, the step ofselecting and rearranging each segment of VH-D-JH is a genetic changethat occurs in the chromosome along with the maturation of B cells. Therearrangement of heavy chain VH-D-JH genes at the antibody gene lociduring B cell differentiation occurs regardless of the presence ofantigens. One B-cell expresses one set of VH-D-JH genes. Derivation fromthe same germline does not mean for the cells to produce the same VHH.

Antibody producing cells are a population of cells each comprising adifferent combination of VH-D-JH genes. When constructing a library ofVHH genes, loss of a portion of the germline-derived antibody genes(i.e., the combinations of VH-D-JH genes) causes a bias in the library.The diversity of VHH genes is not only supported by the mechanism ofrearrangement of VH-D-JH genes. However, since it is extremely difficultto regenerate a lost portion of germline-derived antibody genes viamutations, the importance of constructing a library maintaining theratio of germline-derived antibody genes can be understood easily.Therefore, it is questionable whether the lost part of thegermline-derived genes can be supplemented by attempts to enlarge therepertoire size by artificially introducing mutations to VHH genes.

In the present invention, whether a library maintains the in vivodiversity can be confirmed as follows. Specifically, when a sufficientamount of clones randomly selected from clones constituting a libraryare determined to comprise genes of, desirably 6 VHH subfamilies and atthe same time 15 or more classes, the library is considered to maintainthe in vivo diversity. A sufficient amount of clones used for theanalysis is, for example, 50 clones or more.

The VHH libraries of the present invention allow contamination of genesother than VHH genes. Such contamination is unlikely to become anobstacle for obtaining VHHs, particularly when VH is contaminated andVHHs that bind to difficultly recognized antigenic determinants areobtained. This is because even if VHs coexist in the library, theirpossibility to occupy antigens is low. However, considering that VHHlibraries are screened with the expectation of benefits intrinsic toVHHs, it is preferred to avoid the contamination of genes other than VHHgenes. Normally, 60% or more, preferably 70% or more, and morepreferably 90% or more of the genes of a VHH library of the presentinvention is VHH genes. When the rate of genes other than VHH genes suchas VH increases in the library, under some circumstances, it maydecrease the screening efficiency of VHH genes.

VHH genes can be selectively obtained, for example, throughamplification of the VHH genes by PCR using the aforementioned primersfor VHH gene amplification found by the present inventors. Thusconstructed VHH libraries of this invention has a remarkably highcomponent percentage of VHH genes. A preferable VHH library of thepresent invention comprises VHHs at a rate of, for example, 70% or more,preferably 80% or more, and more preferably 90% or more. Morespecifically, the present invention relates to a VHH library thatcomprises IgG2-derived VHHs at a rate of 90% or more, such as 93% ormore. Furthermore, the present invention also relates to VHH librariescomprising IgG3-derived VHHs at a rate of 95% or more, preferably 95% ormore, and more preferably 99% or more.

-   (3) In the interest of amplifying VHH genes, collecting    amplification products during the progress of exponential    amplification Normally, there is an upper limit to the amount of    products obtainable by gene amplification reactions as represented    by PCR. That is, when the reaction reaches a certain level,    amplification reaction stops proceeding. This means that, regardless    of the amount of template, the amount of amplification products that    are obtainable as a result of an amplification reaction is constant.    Antibody genes exist in vivo as a complex assembly of a variety of    genes. When such an assembly is used as a template for artificial    amplification, the products are often occupied by genes that are    dominant in number. This is because the amplification reaction stops    at the point where the amplification reaction of the dominant genes    reaches the upper limit. When such a phenomenon arises, many of the    repertoires will be lost by gene amplification.

The present inventors found that by collecting amplification productsduring the progress of exponential amplification in the geneamplification reaction, the danger of losing the repertoire through geneamplification could be diminished. While exponential amplification istaking place, all templates can be considered as being amplified atnearly the same probability. Therefore, when amplification products arecollected during that phase, the loss of repertoire during theamplification reaction can be kept down to minimum.

Whether exponential amplification is taking place can be confirmed bymonitoring the amount of gene amplification products. Particularly, whenthe progress of reaction is controlled by the number of reaction cyclesas in PCR, the amount of amplification products is monitored for eachreaction cycle, and the number of cycles necessary for the reaction toreach the upper limit can be elucidated. By performing the amplificationreaction in the presence of intercalators such as SYBR green, the amountof amplification products can be monitored according to the changes influorescence intensity. Alternatively, an aliquot may be obtained fromthe reaction solution and subjected to electrophoresis to visuallyconfirm the amplification products.

For example, under the conditions indicated in the Examples,amplification products can be collected during exponential amplificationby performing PCR with not more than 17 cycles. In PCR, the state ofexponential amplification is determined by various conditions. They areaffected by factors, for example, the amount of template genes, theamount of primers, and the kind and amount of DNA polymerase. Therefore,under conditions different from the Examples, exponential amplificationmay continue even at 17 cycles or more. Whether exponentialamplification is taking place under a certain condition can be confirmedas described above.

When VHH genes are amplified under such conditions, the balance amonggenes in the amplification products reflects the numerical balance amongeach gene in the template. As a result, the possibility of obtainingnumerically dominant genes as well as minor genes increases.

VHH genes obtained under such conditions can be formed into a libraryaccording to any method. To use a library of the present invention inthe screening based on binding affinity, it is advantageous to constructthem as an rgdp library. An rgdp library refers to a replicable geneticdisplay package library. More specifically, it refers to a library thatmaintains genes, and at the same time presents expression products ofthe genes on the surface (genetic display package). A representativergdp library includes a phage library that utilizes the method of phagedisplay. Examples of rgdp libraries include, in addition to the phagelibrary, libraries consisting of transfected cells or ribosomes thatexpress exogenous proteins on their surface.

The phage display method was devised by Smith in 1985 (Smith GP, Science228(4075), 1315-7 (1985)) using a filamentous bacteriophage havingsingle-stranded circular DNA, such as M13 phage. A phage particleconsists of a protein called cp8 and 5 proteins called cp3, bothsurrounding the DNA of the phage; cp8 constitutes the majority of thephage particle and cp3 functions when the phage infects E. coli. A phagedisplay system is a system wherein a gene is constructed so that theyencode a polypeptide in the form fused with cp3 or cp8, and the fusedprotein is expressed on the surface of the phage particle. Phageparticles carrying binding proteins on their surfaces can beconcentrated using the binding activity with their ligands. Such amethod of concentrating the DNA of interest is called “panning”. DNAencoding a protein having required binding activity is packaged in theconcentrated phage particles. Utilizing a filamentous phage as describedabove, a system allowing very efficient screening based on bindingactivity and cloning of DNA was realized (Published Japanese Translationof International Publication No. Hei 5-508076). In the interest of thefilamentous phage libraries, a method allowing expression as Fabmolecules has been reported (Published Japanese Translation ofInternational Publication No. Hei 6-506836). In this report, the fusionof the variable regions was attempted via a method deleting theN-terminus of cp3 and such.

VHH genes obtained as described above can be made into a phage library,for example, as follows. First, amplification products of VHH genes aretreated with restriction enzymes. 5′ primers, oligonucleotides havingthe nucleotide sequences of-SEQ ID NOs: 1 to 6, indicated in theExamples comprise a SfiI site. The obtained fragments are inserted intoexpression vectors for VHH gene transfer that enables expression offusion proteins with a phage protein cp3 or cp8 is used as the phageprotein, the fusion partner with the VHH genes. In the expressionvectors, restriction enzyme sites are prepared to introduce the VHH genefragments. For example, SfiI-AscI treated fragments of the VHH geneamplification products can be introduced into the SfiI-AscI site of thevectors used in the Examples.

To incorporate VHH genes, Cre recombinase can also be used.Specifically, using primers attached with LoxP sequence, VHH geneamplification products carrying LoxP sequences on both ends areobtained. Then, when LoxP sequence is also placed at the integrationsite on the expression vector, the amplification products and the vectorcan be recombined by the action of Cre recombinase.

A vector comprising a gene encoding a phage surface protein such as cp3and cp8, and a site for VHH gene integration located so as to expressthe VHH gene as a fusion protein with the phage surface protein is usedas the expression vector for VHH gene transfer. For example, a SfiI/AscIsite indicated in the Examples can be used as the site for VHH geneintegration. According to the analysis by the present inventors,recognition sequences of these restriction enzymes could not be found incamel VHH genes. Therefore, the combination of restriction enzymes,SfiI/AscI, is useful to construct camel VHH gene libraries.

Furthermore, to use a culture supernatant of a host microorganisminfected with a phagemid as a sample for screening, an expression vectorcomprising a promoter and a signal sequence that function in the hostmicroorganism is used. For example, when using E. coli as a host, aphagemid vector for filamentous phage inserted with a signal sequence,such as pelB sequence, can be used. Examples of expression vectorsuseful for constructing the libraries of the present invention includeexpression vector pFCA-10 indicated in the Examples.

Phage particles expressing a VHH on their surface can be produced bytransfecting a VHH gene-integrated expression vector along with a helperphage into a host, and expressing both of them. By collecting thesephage particles, a phage library of the present invention can beobtained.

The phrase “helper phage” refers to a phage that infects bacteriatransfected with the aforementioned expression vector to provide phagecomponents and produces a phage having a phage surface protein encodedby the expression vector. VHHs will exist on the surface of the phageparticle due to the use of a fusion protein derived from the expressionvector.

The VHH phage libraries of the present invention find use in screeningVHHs by the panning method. The panning method utilizes the bindingaffinity of VHHs for a substance of interest to select phages thatexpress the VHHs. More specifically, first, a phage library expressingVHHs is contacted with a substance of interest, and phages that bind tothis substance are collected. The collected phages are amplified asneeded, and are repeatedly contacted with the substance of interest andcollected. Using the panning method, phages expressing VHHs havingbinding affinity for the substance can be obtained. Genes encoding VHHsare packaged in the phages. Therefore, obtaining phages means nothingbut simultaneously obtaining genes encoding VHHs.

The VHH libraries of the present invention are libraries with extremelylarge repertoire sizes that exceed the in vivo diversity of VHHs.Therefore, the use of a VHH library of this invention allows to readilyselect VHHs with functions that are difficult to obtain by conventionalimmunological manipulations. For example, as already mentioned,obtaining tetrameric IgG-type antibodies that regulate the activity ofan enzyme is often difficult. However, antibodies of VHH constitutingdimeric immunoglobulins that regulate enzyme activity may be easilyobtained. For example, the Examples successfully employ the VHHlibraries of the present invention to yield a plurality of VHHs havingthe function to regulate the activity of a given enzyme. Thus, thepossibility of a non-immunized camel-derived VHH or VH library toprovide a plurality of VHH genes having the function to regulate theactivity of a given enzyme serves as an index to evaluate the diversityof the library.

As described above, the VHH libraries or VH libraries of the presentinvention are useful for obtaining VHHs having the function to regulateenzyme activity. Specifically, VHHs or VHs that have the function toregulate enzyme activity can be obtained via the use of an enzymemolecule or a fragment thereof as the aforementioned substance ofinterest. The fragment of the enzyme molecule can be obtained byfragmenting the enzyme. The enzyme molecule or fragment thereof used forthis purpose may be a fusion protein with another protein.Alternatively, a partial sequence of the gene encoding the enzyme can beexpressed to use its expression product as the fragment. When theobjective is to obtain VHHs or VHs having the function to regulateenzyme activity, the use of a fragment comprising the active site of theenzyme is advantageous. Methods for identifying enzyme active site arewell known in the art. Through further confirmation of the action onenzyme molecules, VHHs or VHs selected by the panning method can beultimately evaluated for their function to regulate the enzyme activity.More specifically, VHHs or VHs to be evaluated are contacted with theenzyme molecule, and when the enzyme activity changes compared to thatwithout this contact, those VHHs or VHs are confirmed to have thefunction to regulate the enzyme activity. In the present invention, thefunction to regulate enzyme activity includes inhibition and promotionof enzyme activity.

Genes encoding VHHs or VHs obtained based on the present invention canbe translated into immunoglobulins or immunoglobulin fragments usingappropriate expression systems. Specifically, the present inventionrelates to a method for producing an immunoglobulin that comprisecamelid-derived VHH as the variable region, or fragments thereof, whichcomprises the steps of following (1) to (3). Alternatively, using VHinstead of VHH, an immunoglobulin comprising VH can be similarlyproduced.

-   (1) Obtaining a gene encoding VHH that has the binding activity    towards a substance of interest by the above-mentioned method;-   (2) producing a VHH expression vector by integrating the obtained    gene encoding VHH to a vector that is expressible in a host cell;    and-   (3) introducing the VHH expression vector into the host cell, and    collecting VHH-comprising proteins from the culture.

Genes encoding VHHs obtained from an rgdp library based on the presentinvention can be collected from VHH expression vectors. For example, theVHH genes of interest can be amplified via PCR using primers used forlibrary construction and expression vectors as templates. Alternatively,when the objective VHH clones can be yielded in large quantities, VHHgene fragments can be excised by restriction enzyme treatment.

The selected VHH genes can be made into complete dimeric immunoglobulinsby linking them with a gene encoding the constant region. For example,when utilizing the impairment effect of IgGs on cells and viruses, IgGscomprising the constant region are more advantageous. A safepharmaceutical formulation is provided by forming a chimeric antibodythrough the combination of the human constant region. On the other hand,for in vitro diagnostic agents and industrial applications, VHHs can beused as they are.

Alternatively, appropriate tags can be attached to VHHs to form fusionproteins. His tags may be used as the tags. His tagged VHHs can beeasily purified using nickel column and such. Furthermore, VHHs can beexpressed as fusion proteins with a heterogeneous protein, such as GFPor RFP (Morino et al., J. Immunol. Methods 257, 175-184 (2001)).

To express VHH genes as VHHs, or as fusion proteins comprising VHHs, anyvector may be used as the expression vector. For example, vectors suchas pCANTAB 5E (Amasham), pTZ19R, and pTZ18R are useful for theexpression of immunoglobulins. Methods for constructing these vectorsare well known in the art. Expression vectors carrying VHH genes can betransformed into hosts that are appropriate for each vector. Forexample, the aforementioned expression vectors can be transformed intoDH12s, TG1, HB2151, and such, to express VHHs. VHHs and immunoglobulinscomprising VHHs that are obtained according to the present inventionhave a variety of uses.

For example, development of diseases due to viruses, bacteria,parasites, or other pathogenic factors can often be avoided byinhibiting enzyme activity of the pathogenic factor or recognition oftarget molecules by the pathogenic factors via the binding ofimmunoglobulins. Furthermore, through the binding of immunoglobulins tothe active (toxic) site of toxic substances, deleterious effects of thesubstance can be avoided.

Not only a function to inhibit enzyme activity but also a function toenhance enzyme activity can be expected from camel antibody VHs and VHHsobtained by the present invention. Camel antibody VHs and VHHs havingsuch functions are also expected to suppress exacerbation of symptoms,alleviate pathologic pains, or cure integrated dysfunction of complexedenzymatic or physiological processes due to the disorder of enzymefunction or protein recognition.

Furthermore, enzyme inhibitory antibodies obtained by the presentinvention can be used to evaluate enzyme activity. Specifically, when anenzyme inhibitory antibody inhibits a certain enzyme activity in asample, the existence of an enzyme that is inhibited by the enzymeinhibitory antibody can be elucidated. By correlating the added amountof the enzyme inhibitory antibody and the inhibition level, the level ofenzyme activity in the sample can be quantitatively evaluated.

In addition, antibodies obtained by the present invention having thefunction to enhance enzymatic activity find use in detecting enzymeactivity. Specifically, a detection system wherein a small amount ofenzyme activity is amplified with an antibody that has the function toenhance the enzymatic activity may be constructed. The present inventionprovides testing and measuring kits for enzyme activity, and diagnostictechniques that use the antibodies obtained by the present invention.

Furthermore, the antibodies obtained by the present invention having thefunction to regulate enzyme activity can be used in industrial enzymaticreactions. For example, the yield of an objective product of anenzymatic reaction system can be increased by colocalizing antibodiesthat promote enzyme activity. Conversely, the yield of products can bealso increased by colocalizing antibodies that suppress enzyme activityconsuming the objective product of interest in the enzymatic reactionsystem. Moreover, the purity of products is expected to be increased viathe suppression of enzyme activity generating undesirable by-products.

All references cited herein are incorporated by reference into thepresent application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of the vector for camel antibody library(pFCA-10) and known vectors (pscFvCA-E8VHd vector andscNcopFCAH9-EBVHdVLd vector) used for its construction.

FIG. 2 shows the nucleotide sequence of the expression vector pFCA-10for camel VHH integration and the amino acid sequence encoded by itsprotein-coding region.

FIG. 3 shows the result of monitoring the amount of amplificationproducts in each reaction cycle of PCR. Control DNA stock solution, its10-fold dilution, or 100-fold dilution was used as the template. Thevertical axis shows the amount of DNA (μg/mL) on a logarithmic scale andthe horizontal axis shows the number of PCR cycles.

FIG. 4 is a set of photographs showing the result of electrophoresis ofamplification products at each reaction cycle of PCR using camelantibody cDNA as the template. The names of the 5′ primers are indicatedunder the photographs. From the left for each of the primers,amplification products of the 15th, 17th, 19th, and 21st cycle wereeledtrophoresed. The results of using primers for IgG2 and primers forIgG3 as 3′ primers are shown in the upper and lower photographs,respectively.

FIG. 5 shows the result of confirming VHH-cp3 expression in ElectroMAX™DH12S (GIBCOBRL) transfected with camel VHH gene (IgG2)-integratedexpression vectors. The vertical axis shows the absorbance (OD 492 nm),and the horizontal axis shows the sample number.

FIG. 6 shows the result of confirming VHH-cp3 expression in ElectroMAX™DH12S (GIBCOBRL) transfected with camel VHH gene (IgG3)-integratedexpression vectors. The vertical axis shows the absorbance (OD 492 nm),and the horizontal axis shows the sample number.

FIG. 7 shows the results of ELISA confirming the responsiveness of VHH(IgG2) clones to GST. The vertical axis shows the absorbance (OD 492nm), and the horizontal axis shows the sample number.

FIG. 8 shows the results of ELISA confirming the responsiveness of VHH(IgG3) clones to GST. The vertical axis shows the absorbance (OD 492nm), and the horizontal axis shows the sample number.

FIG. 9 shows the result of measuring the inhibitory effect of anti-GSTVHHs on GST activity. The vertical axis shows the residual activity (%)of GST, taking the enzyme activity in the absence of VHH action as 100,and the horizontal axis shows the concentration (μM) of added VHH.

FIG. 10 shows the result of examining the competitive effect of anti-GSTVHHs on glutathione. The vertical axis shows the reciprocal of theamount of change in absorbance per minute, and the horizontal axis showsthe reciprocal of CDNB concentration.

FIG. 11 shows the result of examining the competitive effect of anti-GSTVHHs on CDNB. The vertical axis shows the reciprocal of the amount ofchange in absorbance per minute, and the horizontal axis shows thereciprocal of glutathione concentration.

FIG. 12 shows the result of ELISA confirming the responsiveness of VHHclones to LDH in the third screening cycle. The vertical axis shows themeasured value at OD 492 nm in ELISA, and the horizontal axis shows thesample number.

FIG. 13 shows the result of ELISA confirming the responsiveness of VHHclones to LDH in the fourth screening cycle. The vertical axis shows themeasured value at OD 492 nm in ELISA, and the horizontal axis shows thesample number.

FIG. 14 shows the results of measuring the effect of anti-LDH VHHs onLDH activity. The vertical axis shows the measured value at OD 492 nm inELISA, and the horizontal axis shows the elapsed time (minutes) from thestart of the enzyme reaction.

FIG. 15 shows the result of measuring the VHH concentration dependenceof the LDH activity inhibitory action by anti-LDH VHHs. The verticalaxis shows the residual activity (%) of LDH taking the enzyme activityin the absence of VHH action as 100, and the horizontal axis shows theconcentration of added VHH (μM).

FIG. 16 shows the nucleotide sequence of the insert inscNcopFCAH9-E8VHdVLd and the amino acid sequence encoded thereby(continued to FIG. 17).

FIG. 17 shows the nucleotide sequence of the insert inscNcopFCAH9-E8VHdVLd and the amino acid sequence encoded thereby(continued from FIG. 16).

FIG. 18 shows the result of ELISA confirming the responsiveness of VHH(IgM) clones to β-Gal. The vertical axis shows the absorbance (OD 492nm) and the horizontal axis shows the sample number.

FIG. 19 shows the nucleotide sequence of the insert in pscFvCA-E8VHd andthe amino acid sequence encoded thereby (continued onto FIG. 20).

FIG. 20 shows the nucleotide sequence of the insert in pscFvCA-E8VHd andthe amino acid sequence encoded thereby (continued from FIG. 19).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in detail below with referenceto the Examples.

EXAMPLE 1 Construction of Camel Antibody Library

1-1. Confirmation of Restriction Enzyme Sites in Camel Germline andPrimer Designing

In the following Examples, New England biolabs or buffers produced bythis company will be indicated as NEB. The absence of sites cleavablewith SfiI or AscI restriction enzyme used for subcloning into a vectorwas confirmed in the germlines of camel VH or VHH by referring to areference (Nguyen et al., EMBO J. 19(5), 921 (2000)). Six kinds ofprimers for the N-terminus of the V region covering the germlines of VHand VHH were constructed. The nucleotide sequences of the primers areshown in SEQ ID NOs: 1 to 6. (1) VHH3a (SEQ ID NO: 1) GTCCTCGCAACTGCG GCC CAG CCG GCC ATG GCC GAG GTG CAG CTG GTG GAG TGT GG 2) VHH-germF1(SEQ ID NO: 2) GTCCTCGCAACT GCG GCC CAG CCG GCC ATG GCC CAG GTR CAG CTGGTG GAG TCT GG 3) VHH-germF2 (SEQ ID NO: 3) GTCCTCGCAACT GCG GCC CAGCCG GCC ATG GCC CAG GTA AAG CTG GAG GAG TCT GG 4) VHH-germF4 (SEQ ID NO:4) GTCCTCGCAACT GCG GCC CAG CCG GCC ATG GCC GAT GTG CAG CTG GTG GAG TCTGG 5) VHH-germF5 (SEQ ID NO: 5) GTCCTCGCAACT GCG GCC CAG CCG GCC ATG GCCGCC GTG CAG CTG GTG GAT TCT GG 6) VHH-germF6 (SEQ ID NO: 6) GTCCTCGCAACTGCG GCC CAG CCG GCC ATG GCC GCG GTG CAG CTG GTG GAG TCT GG * SfiI sitesare underlined.

Next, C-terminal primers of the V region were designed.

Considering the convenience during screening, VHs (IgG1) that may causeaggregation were removed as possible from the camel library constructedherein. Therefore, IgG1 was removed and IgG2 and IgG3 were selectivelyincluded in the library. For this purpose, sequences absent in IgG1 butexisting in IgG2 and/or IgG3 alone had to be used as primers.

Accordingly, the present inventors focused on the differences in thesequences of the hinge regions existing on the C-terminal side of the Vregion. The previously obtained hinge region of camel IgG1 gene (PCRproduct of 800 bp) was analyzed and compared with IgG2 and IgG3. As aresult, the amino acid sequences in this region showed the followingdifferences. tetrameric camel antibody: IgG1 hingeEPHGG                          CPCPKCP (SEQ ID NO: 7) dimeric camelantibody: IgG2 hinge EP KI PQPQPKPQPQPQPQPKPQPKPEPE CTCPKCP (SEQ ID NO:8) IgG3 hinge GT N EV                        CKCPKCP (SEQ ID NO: 9)

Based on these results, primers that selectively amplify IgG2 and IgG3were designed by determining the N-terminal nucleotide sequence of thehinge regions existing on the C-terminal side of the V region. Thenucleotide sequences of the C-terminal (3′-side) primers that were thusdetermined are shown in SEQ ID NO: 10 (IgG2-LB1) and SEQ ID NO: 11(IgG3-LB2). IgG2-LB1 (SEQ ID NO: 10): AA G GCG CGC CCC TTG GGG TAT CTTGGG TTC TG IgG3-LB2 (SEQ ID NO: 11): AA G GCG CGC CCC TGA TAC TTC ATTCGT TCC TGA VG AG1-2. Cloninq Vector for Library

First, an expression vector inserted with a stuffer sequence into theSfiI-AscI site wherein a VHH gene is integrated was constructed. Thestructures of pscFvCA-E8VHd vector, scNcopFCAH9-E8VHdVLd vector, andexpression vector pFCA-10 for camel VHH integration, all used for vectorconstruction, are shown in FIG. 1. In addition, the nucleotide sequenceof pFCA-10 and the amino acid sequence encoded by its protein-codingregion are shown in FIG. 2. The vectors were designed so as not toexpress cp3, when the restriction enzyme failed to cleave the vector orthe antibody gene had not been integrated. scNcopFCAH9-E8VHdVLd andpFCA-10 were constructed as described below.

1-2-1. Construction of ScNcopFCAH9-E8VHdVLd

Three μg (3 μL) of pFCAH9-E8d (see WO 01/62907) was mixed with 3 μL ofBstPI (3 U/μL), 5 μL of 10×H buffer, and 39 μL of distilled water (DW),and restriction enzyme treatment was performed at 37° C. for 2 hr. Afterthe treatment, the precipitate obtained by ethanol precipitation wasdissolved in 10 μL of TE buffer. One μL of SacI (10 U/μL), 5 μL of 10×Lbuffer, and 34 μL of DW were mixed for restriction enzyme treatment at37° C. for 2 hr, and then a 4.7 kb fragment was collected by agarose gelelectrophoresis. The collected fragment was subjected to ethanolprecipitation and adjusted to 10 μL (pFCAH9-E8d BstPI-SacI fragment).

Meanwhile, 5 μL of linF primer (100 pmol/μL) and 5 μL of linR primer(100 pmol/μL) were mixed. The mixture was heated at 94° C. for 5 min,incubated at 80° C. for 5 min, 70° C. for 5 min, and then left at roomtemperature for 30 min to be annealed. Two μL of this solution, 1 μL ofthe pFCAH9-E8d BstPI-SacI fragment obtained above, 1.5 μL of 10×ligationbuffer, 9.5 μL of DW, and 1 μL of T4DNA ligase were mixed and reacted at16° C. for 16 hr. After the reaction, the mixture was concentrated to 3μL by ethanol precipitation, and 1.5 μL thereof was used to transform 20μL of E. Coli DH12S competent cells by electroporation. The plasmid ofthe obtained clone was extracted, its nucleotide sequences wasconfirmed, and the plasmid was dubbed scNcopFCAH9-E8VHdVLd. A schematicillustration of the structure of scNcopFCAH9-E8VHdVLd is shown in FIG. 1(top). Furthermore, the nucleotide sequence of the insert region ofscNcopFCAH9-E8VHdVLd and the amino acid sequence encoded thereby areshown in FIGS. 16 and 17. In addition, the nucleotide sequence of theinsert region of pscFvCA-E8VHd and the amino acid sequence encodedthereby are shown in FIGS. 19 and 20. linF primer (SEQ ID NO: 36)GTCACCGTCTCGAGAGGCGGTGGCGGATCAGGTGGCGGTGGAAGTGGCGGTGGTGGGTCCATGGCCGACATCGAGCT linR primer (SEQ ID NO: 37)CGATGTCGGCCATGGACCCACCACCGCCACTTCCACCGCCACCTGATCCG CCACCGCCTCTCGAGACG1-2-2. Construction of Vector (pFCA-10) for Camel Antibody Library

Two μg of scNcopFCAH9-E8VHdVLd vector (10 μL), 10 μL of 10×M buffer, 78μL of DW, and 2 μL of HindIII (12 U/μL; Takara) were mixed and incubatedat 37° C. for 2 hr, concentrated by ethanol precipitation, and dissolvedin 10 μL of TE. Subsequently, 10 μL of 10×NEB4 buffer (supplied withAscI), 78 μL of DW, and 2 μL of AscI (10 u/μL; NEB) were added thereto,incubated at 37° C. for 2 hr, concentrated by ethanol precipitation, andelectrophoresed on agarose gel to collect a fragment of interest (3.7kb). The fragment was purified using GENECLEAN II Kit (Funakoshi),concentrated by ethanol precipitation, and dissolved in 10 μL of 1/10TE.

Next, 0.1 μg of pscFvCA-E8VHd vector (5 μL), 1 μL of M13RV primer (100pmol/μL, 5′-AACAGCTATGACCATG-3′; SEQ ID NO: 12/Sawady Technology), 1 μLof XhoAsc primer (100 pmol/μL,5′-CGACTGAAGGCGCGCCCCTCTCGAGACCCTGACCGTGGTGCC-3′; SEQ ID NO: 13/SawadyTechnology), 10 μL of 10×buffer #1 (supplied with KOD), 10 μL of dNTPmix (supplied with KOD), 4 μL of 25 mM MgCl₂, 68 μL of DW, and 1 μL ofKOD polymerase (2.5 u/μL; TOYOBO) were mixed on ice, 2 drops of mineraloil were added and warmed at 94° C. for 2 min. Subsequently, treatmentat 94° C. for 1 min, 55° C. for 2 min, and 72° C. for 1 min was repeatedfor 25 cycles. The obtained PCR product was confirmed by agarose gelelectrophoresis. Then, a band near 400 bp was cut out, purified usingGENECLEAN II Kit (Funakoshi), ethanol precipitated, and then suspendedin 10 μL of TE.

10 μL of 10×M buffer, 78 μL of DW, and 2 μL of HindIII (12 U/μL; Takara)were mixed to the above DNA solution. The mixture was incubated at 37°C. for 2 hr, and concentrated by ethanol precipitation, and thendissolved in 10 μL of TE. Subsequently, 10 μL of 10×NEB4 buffer(supplied with AscI), 78 μL of DW, and 2 μL of AscI (10 U/μL; NEB) weremixed thereto, incubated at 37° C. for 2 hr, concentrated by ethanolprecipitation, and then electrophoresed on agarose gel. The band near340 bp was collected, purified using GENECLEAN II Kit (Funakoshi),concentrated by ethanol precipitation and then dissolved in 10 μL of1/10 TE. To a half of this solution, 5 μL, 2 μL of the HindIII-AscIfragment of the scNcopFCAH9-E8VHdVLd vector, 2 μL of 10×ligation buffer,2 μL of 10 mM ATP, 8 μL of DW, and 1 μL of T4 DNA ligase were added andmixed, and the mixture was incubated at 16° C. for 16 hr. This wasethanol precipitated and dissolved in 3 μL of 1/5 TE. Then, half thereofwas suspended in 20 μL of ElectroMAX™ DH12S (GIBCO BRL) competent cellsto achieve transformation via electroporation under followingconditions: Electroporator BRL Cell-Porator (Cat. series 1600) setupconditions; voltage booster 4 kΩ capacitance 330 μF DC volts Low Ωcharge rate Fast

Twelve obtained transformants were cultured in LBGA at 30° C. for 18 hr,plasmids were extracted using KURABO DNA Isolation System PI-50, andtheir nucleotide sequences were confirmed. The sequences were determinedby the dideoxy method using M13 Reverse fluorescent primer (cat. No.LIC-4000-21B; ALOKA), Thermosequence kit (Amersham Pharmacia), and ALOKAL1-COR4200L(S)-2. As a result, all sequences were as designed. One ofthem (No. 1) was prepared from 400 mL of culture medium by the alkalinemethod, and then purified by CsCl density gradient ultracentrifugationto yield 210 μg of plasmid. It was named pFCA-10, and used as a vectorfor camel antibody library.

1-3. Preparation of Total RNA from Frozen Spleen (GuanidineUltracentrifugation Method)

Approximately 9 g of frozen spleen from dromedaries (Camelusdromedarius) was finely crushed with a hammer. After measuring theweight, 45 ml of GTC solution (450 μL of 2-mercaptoethanol added rightbefore use to 4.0 M guanidinethiocyanate and 0.1 M Tris-HCl (pH 4.5))was added immediately, and was further fragmented in an ice-cold nisseiExcel Auto Homogenizer at 17,000 rpm for 2 min. Next, it was completelyhomogenized with a Teflon homogenizer to remain connective tissue debrisalone. The homogenate solution was filtered through gauze.

0.23 g of N-lauroylsarcosin sodium salt was added as a powder to thefiltered solution, and was dissolved well. DNA therein was fragmented byshearing force due to passages of the solution through needles attachedto a syringe, which involved 3 passages through 18G, 5 passages through21G, and 5 passages through 22G needles.

The precipitates were removed by centrifugation at 5,000 rpm for 10 minat. room temperature. The solution was approximately 36 ml. HITACHICentrifuge Ware 13PA tube was washed with diothylpyrocarbonate (DEPC)treated water, 4 mL of CsCl solution (5.7 M CsCl, 0.01 M EDTA) wasplaced therein, and 6 ml of supernatant was layered without disturbingthe interface. Ultracentrifugation was performed on HITACHI HIMAC55P-72using RP40T-740 rotor at 30,000 rpm for 20 hr. (Except TES solution (10mM Tris-HCl, 5 mM EDTA, 1% SDS), reagents used for following steps wereall treated with DEPC, and operations were carried out with utmost careto avoid contamination by ribonucleases.)

After centrifugation, the upper layer was carefully removed (Pasteurpipettes were frequently exchanged) When the remaining upper layerbecame approximately 0.5 mL, the tube was inverted to discard theremaining solution. Using a heated razor blade, near 1 cm from thebottom of the tube containing RNA precipitate was cut, and theprecipitate was rinsed with 75% ethanol and air-dried. The precipitatewas dissolved in TES solution, 1/10 volume of 3M sodium acetate (pH5.2)and 2.5 volume of ethanol were added for precipitation, and theprecipitate was stored at −80° C. until use.

Total RNA was obtained by guanidine ultracentrifugation from spleens of22 camels according to similar steps described above. mRNA was preparedfrom each of thess total RNAs using commercially available oligo-dTcolumn kit (Pharmacia mRNA Purification Kit (oligo-dT column method)).The yield of mRNA was about 1% of the total RNA.

1-4. Construction of Heavy Chain Variable Region Antibody Libraries forVHH Selection

1-4-1. Preparation of cDNA

One μg each of mRNA derived from each camel was mixed to form a librarytemplate. cDNA was prepared by reverse transcriptase reaction on themRNA using a random primer (N6, Gibco BRL) and following theinstructions for Superscript (Gibco BRL).

The incorporation of [α-³²P]dCTP was measured at real time to reveal a[α-³²P]dCTP incorporation of 2.4% and a transcription rate of 15.8%.Calculating from the incorporation rate, 3.3 μg of cDNA was obtainedfrom 22 μg of mRNA.

1-4-2. PCR for Construction of Libraries, and Collection of VHH GeneFragments

Separate libraries were constructed for IgG2 and IgG3, respectively, asheavy chain variable region antibody libraries for VHH selection. Toconstruct libraries reflecting the germline ratio expressed in 22camels, PCR amplification process was monitored and amplificationproducts were collected in the exponential phase. The PCR amplificationprocess was stopped at every few cycles to monitor the DNA level byquantification. Even for diversified fragments, a group of genefragments accurately reflecting the repertoire before the amplificationshould be obtained by collecting them while exponential geneamplification is taking place.

Stock solution of control DNA constructed by ligating a camel antibodygene into Bluescript vector, 10-fold diluted and 100-fold dilutionsthereof were used as templates to confirm gene amplification (FIG. 3).Here, VH3a primer (SEQ ID NO: 1) was used as the N-terminal primer, and“J primer” (5′-AA GGCGCGCCCC TGA VGA GRY GGT GAC YHG-3′; mixednucleotides as, V: ACG, R: AG, Y: CT, and H: ACT; SEQ ID NO: 42)constructed based on the J gene was used as the C-terminal primer.Similar experiments were performed, wherein the N-terminal primer waschanged to VHH-germ F1 (SEQ ID NO: 2) or VHH-germF2 (SEQ ID NO: 3) andcontrol DNA stock solution was used as a template. These results arealso shown in FIG. 3 as primer land primer 2, respectively.

The reaction was stopped at every few cycles and a portion of thereaction solution was sampled. After staining with Picogreen (GibcoBRL), DNA was quantified using a fluorophotometer. At stages where theDNA concentration is low, such as up to the 20th cycle of the 10-folddilution or 100-fold dilution, the apparent amplification rate seemedlow in certain cases due to measurement noise. However, up toapproximately 27 cycles, exponential amplification could be confirmed.

Next, cDNA actually obtained in 1-4-1 and primers of SEQ ID NOs: 1 to 6were used for gene amplification. The reaction was stopped at 15th,17th, 19th, and 21st cycles and 2 μL of each of the reaction solutionswas electrophoresed. As a result, the reaction did not reach saturationat least up to the 19th cycle (FIG. 4).

According to the above-mentioned results, 17 cycles of PCR reactionswere carried out on the same amount of cDNA using combinations of 6types of 5′ primers with 2 types of 3′ primers (IgG2 LB1 or IgG3 LB2).The PCR conditions were as follows: LA Taq (Takara) 0.5 μL 10× LA buffer(Takara; supplied with LA Taq) 10 μL 25 mM MgCl₂ (Takara; supplied withLA Taq) 10 μL dNTP (Takara; supplied with LA Taq) 16 μL sterilizedMilliQ water 61.5 μL Template cDNA 1 μL 5′ primer (100 pmol/μL) 0.5 μL3′ primer (100 pmol/μL) 0.5 μL

Any one of VH3a (SEQ ID NO: 1), VHH-germF1 (SEQ ID NO: 2), VHH-germF2(SEQ ID NO: 3), VHH-germF4 (SEQ ID NO: 4), VHH-germF5 (SEQ ID NO: 5),and VHH-germF6 (SEQ ID NO: 6) was used as the 5′ primer. Furthermore,either IgG2LB1 (SEQ ID NO: 10) or IgG3LB2 (SEQ ID NO: 11) was used asthe 3′ primer. PCR was performed on all combinations of these primers (6types of 5′ primers×2 types of 3′ primers=12 combinations). 50 μL ofmineral oil (SIGMA) was layered on the above-mentioned reactionsolution-in 110 of 0.5-mL tubes when VHH-germF1 was used as the 5′primer, and in 55 tubes in other cases. After incubating the solution at94° C. for 3 min, 17 cycles of amplification at 94° C. for 1 min, 61° C.for 2 min (59° C. when using IgG3LB2), and 72° C. for 1 min wereperformed. PCR amplification fragments of 6 types of IgG2, or 6 types ofIgG3 were mixed respectively and electrophoresed. The bands comprisingthe fragment of interest were cut out and collected using QIAEXII(QIAGEN). For IgG3 and IgG2, 48 μg and 25 μg of PCR fragments werecollected, respectively.

1-4-3. Insertion of PCR-Amplified Gene Fragments into Library Vector

Enzyme fragmentation and ligation conditions are shown below.

(1) SfiI (NEB) cleavage and TsAP (Gibco BRL) treatment of library vectorpFCA-10 vector 100 μg NEB No. 2 200 μL 10× BSA (Takara) 200 μL SfiI 10U/μL 100 μL TsAP 1 U/μL 20 μL

adjusted to 2000 μL using sterilized MilliQ water

Reaction solution with the above-mentioned composition was layered withmineral oil and was reacted at 50° C. for 5 hr. Successively, followingcomponents were further added for TsAP treatment. NEB No. 2 10 μL 10×BSA (Takara) 10 μL DW 40 μL TsAP 1 U/μL 40 μL

After reacting at 65° C. for 30 min, stop solution was added to thereaction solution and was heated at 65° C. for 20 min to inactivate theenzymes. The mixture was treated twice with phenol-chloroform and thenonce with chloroform. The resulting solution was concentrated to 1 mL bybutanol, 210 μg of glycogen was added, and then ethanol precipitated tocollect 105 μg of vector.

Subsequently, by the following operation, PCR amplification products ofIgG2 or IgG3 were cleaved with SfiI. PCR fragment 25.0 μg NEB No. 2 50μL 10× BSA (Takara) 50 μL SfiI 10 U/μL 25 μL

adjusted to 200 μL with sterilized MilliQ water The reaction solutionwas layered with mineral oil and reacted at 50° C. for 3 hr.Phenol-chloroform treatment was performed followed by chloroformtreatment and ethanol precipitation. Twenty-two μg of IgG2 and 23.8 μgof IgG3 were collected.

(2) Ligation of the SfiI Portions SfiI cleaved fragments of 8 μg IgG2 orIgG3 PCR amplification product SfiI-cleaved library vector pFCA-10 40 μg10 mM DTT 40 μL 10 mM ATP 40 μL 10× ligase buffer (Takara, supplied withT4 DNA Ligase) 40 μL T4 DNA Ligase (Takara) 350 U/μL 40 μL adjusted to400 μL with sterilized MilliQ water

The reaction solution was incubated at 16° C. for 13 hr, and at thispoint, following factors were supplemented. DW 120 μL 10 mM DTT 20 μL 10mM ATP 20 μL 10× Ligase Buffer 20 μL T4 DNA Ligase (Takara) 350 U/μL 20μL

This solution was further reacted at 16° C. for 6.5 hr, subjected tophenol-chloroform treatment, and concentrated to 230 μL with butanol. 70μg of glycogen was added thereto, and subjected to ethanol precipitationto obtain 34.1 μg of IgG2 ligation DNA or 29.7 μg of IgG3 ligation DNA.

(3) AscI Cleavage

The collected IgG2 ligation DNA (34.1 μg), or IgG3 ligation DNA (29.7μg) was treated with a reaction solution with following composition at37° C. for 3 hr. NEB No. 4 61 μL AscI (NEB) 10 U/μL 50 μL

adjusted to 610 μL with sterilized MilliQ water

After the reaction, the solution was subjected to phenol-chloroformtreatment, and concentrated to 200 μL with butanol. 70 μg of glycogenwas added thereto, and subjected to ethanol precipitation to obtain 25μg of IgG2 ligation product or 29 μg of IgG3 ligation product.

(4) Ligation of the AscI Portions

25 μg of collected DNA of the IgG2 ligation product, or 29 μg DNA of theIgG3 ligation product was treated with a reaction solution withfollowing composition. 10 mM DTT 750 μL 10 mM ATP 750 μL 10× ligasebuffer 750 μL T4 DNA Ligase (Takara) 350 U/μL 375 μL adjusted to 7500 μLwith sterilized MilliQ water

After 16 hr of treatment at 16° C., the solution was concentrated to 600μL using CENTRICON YM-10 (10,000 molecular weight cut-off, AmiCon), andsubjected to phenol-chloroform treatment. 70 μg of glycogen was thenadded thereto and subjected to ethanol precipitation to obtain 23.7 μgof IgG2 ligation product or 27.8 μg of IgG3 ligation product.

1-4-4. Confirmation of Library Expression

0.2 μg of the expression vector incorporating camel VHH genesconstructed as described above was transformed into 20 μL of ElectroMAXmDH12S (GIBCOBRL). The culture supernatant of the transformants wassampled to confirm the expression of VHH-cp3. Since pFCA-10 comprisespelB secretion signal sequence, a small amount of VHH-cp3 protein of thephage is produced into the culture supernatant of E. coli transformedwith this vector. Therefore, by monitoring the culture supernatant byELISA, the expression of VHH-cp3 can be confirmed. As a result ofmonitoring the expression of VHH-cp3 in the culture supernatant byELISA, 90% expressed VHH-cp3. The results of monitoring by ELISA areindicated in FIG. 5 (IgG2) and FIG. 6 (IgG3). Specific protocols forELISA were as described below.

The expression was induced by the addition of 1 mM IPTG in the earlystage of exponential growth phase. After 21 hr, the culture wascentrifuged and the collected culture supernatant was used to sensitizeMaxiSorp™. 500-fold-diluted rabbit anti-cp3 antibody was used as theprimary antibody and 10,000-fold-diluted HRP-conjugated anti-rabbit IgG(H+L chain) goat Fab′ was used as the secondary. antibody. Formeasurement of HRP activity, 100 μL of ortho-phenylenediamine andhydrogen peroxide solution were added and reacted for 10 min. Thereaction was quenched by adding 100 μL of 2N sulfuric acid, and theabsorbance at a wavelength of 492 nm was measured. 3a-lib1-4 and Sfi36on the right ends in FIGS. 5 and 6 show the results of frame shiftmutation (negative control).

1-4-5. Transformation of Heavy Chain Variable Region Antibody Libraries(IqG2 and IqG3 Libraries) for VHH-Type Selection

E. coli was transformed via electroporation under following conditionsto introduce phage genes.

Electroporator Electroporator BRL Cell-Porator (Cat. series 1600) setupconditions; voltage booster 4 kΩ capacitance 330 μF DC volts Low Ωcharge rate Fast

For IgG2, 23.7 μg of DNA obtained by ligation was used to transform 2 mLof DH12S cells (0.2 μg each of DNA was electroporated into 20 μL ofElectroMAX™ DH12S (GIBCOBRL)). Aportion of these cells was sampled toestimate the overall number of transformed bacteria as 1.7×1010. Thesecells were stored as glycerol stock, and 20-L scale of phage preparationwas carried out.

Specifically, 100 μg/mL of ampicillin was added to 4.8 L of sterilized2×TY (DIFCO) medium, and the glycerol stock was added to adjust theabsorbance at wavelength 600 nm to the vicinity of 0.3. This solutionwas divided into 16 portions of 300 mL, cultured with shaking in asterilized 5 L flask at 37° C. until the absorbance at 600 nm reached1.0. Three mL of helper phage (M13K07) per flask was added to theculture, and cultured at 37° C. for 1 hr. 900 mL of sterilized 2×TYmedium, 0.9 mL of 100 μg/mL ampicillin, and 1.2 mL of 50 μg/mL ofkanamycin were added to each flask and cultured with shaking at 37° C.for 17 hr.

During the culture, the number of bacteria at the time of helper phageinfection was 5.66x 1011 and infectivity of the helper phage was 75%(there were 4.06x 108 cfu/ml of ampicillin-resistant bacteria and3.05×10⁸ cfu/mL of ampicillin- and kanamycin-resistant bacteria duringhelper phage infection). The goal to obtain 10¹⁰ or more independentclones was achieved.

To collect the phages, the bacterial solution was centrifuged at 10,000rpm for 10 min at 4° C. to collect the supernatant. 4 L of 20%polyethylene glycol/2.5 M NaCl was added to the supernatant and wasgently stirred for approximately 20 min.

This solution was centrifuged at 10,000 rpm for 10 min at 4° C. and theprecipitate was dissolved in 1 L of PBS. 200 mL of 20% polyethyleneglycol/2.5M NaCl was added thereto, and gently stirred for approximately20 min. After centrifugation at 10,000 rpm for 5 min at 4° C., thesupernatant was discarded. The residue was further centrifuged at 10,000rpm for 1 min at 4° C., and the precipitate was collected. Theprecipitate was dissolved in 20 mL of PBS containing 0.05% NaN₃ to forma library phage solution.

Next, the titer of the collected phage was checked as follows.Specifically, 10⁶, 10⁷, and 10⁸-fold dilution of the phage solution wasprepared with PBS, 10 μL each was used to infect 990 μL of DH12S cells,and the cells were cultured at 37° C. for 1 hr. 100 μL of the culturewas seeded on LBGA plate, cultured at 30° C. for 18 hr, and the stocksolution titer was estimated from the colony count. As a result, 20 mLof library phage having a titer of 3.73×10¹³ CFU/mL was obtained.

For IgG3, 27.8 μg of DNA obtained by ligation was used to transform 2 mLof ElectroMAX™ DH12S (GIBCOBRL) by electroporation (0.2 μg each wasintroduced into 20 μLof ElectroMAX™ DH12S (GIBCOBRL)) A portion of thecells was sampled to estimate the overall number of transformed bacteriaas 1.1×10¹⁰. The preparation was carried out similarly to IgG2.

During culturing, the number of bacteria at the time of helper phageinfection was 8.64×10¹¹ and infectivity of the helper phage was 78%(4.64×10⁸ cfu/mL of ampicillin-resistant bacteria and 3.63×10⁸ cfu/ml ofampicillin- and kanamycin-resistant bacteria during helper phageinfection). 10¹⁰ or more independent clones, which were the targetamount, were obtained.

These cells were stored as glycerol stock, and a 20-L scale phagepreparation was prepared from the glycerol stock. As a result, 20 mL oflibrary phage having a titer of 4.26×1013 cfu/mL was obtained.

1-4-6. Genetic Sequence Confirmation of the Libraries

VHH clones constituting the libraries were randomly selected todetermine their nucleotide sequences. The nucleotide sequences weredetermined according to the method mentioned in 2-6-2. The amino acidsequences encoded by the determined nucleotide sequences were analyzed.When the analyzed amino acid sequence was in frame and its frameworkregion showed significant homology to that of a known VHH gene, theamino acid sequence was determined to be normal. Actually, no sequencewith considerably low framework homology could be found. Thus, thosewith frame shifts or insertion of stop codon were determined to beabnormal. As a result, for IgG2, the genetic sequences of 87% of theclones were normal. Among those with normal sequences, 92.3% hadhydrophilic amino acids as the 44th and 45th amino acids that interactwith the L chain, i.e., encoded VHHs.

For IgG3, the sequences of 95.8% of the clones were normal. 96% of thenormal clones had hydrophilic amino acids as the 44th and 45th aminoacids that interact with L chains, i.e., encoded VHHs. Considering thatthe ratio of VHs and VHHs in germline genes is 1:1, VHHs were includedin the libraries with very high selectivity.

Thus, libraries having excellent expression rate and high VHH ratio wereconstructed.

EXAMPLE 2 Analysis of VHH Genes

In Example 1, oligonucleotides comprising the nucleotide sequences offollowing SEQ ID NOs were used as primers. The origin of VHH amplifiedby each of the combinations of primers is summarized below.

5′-side (N-terminal). 3′-side (C-terminal) origin of amplified VHH SEQID NOs: 1-6 SEQ ID NO: 10 IgG2 SEQ ID NOs: 1-6 SEQ ID NO: 11 IgG3

Fifty or more clones were randomly selected from each library to analyzetheir nucleotide sequences. Examples of analyzed results are shown inTable 1. Each cell of the Table corresponds to a single class. In Table1, subfamilies are classified based on the position of cysteineresidues, and furthermore, within the subfamilies, classes are groupedbased on the length of CDR2 and CDR1. The determination of CDR wasperformed according to a known method (Kabat et al., Sequence ofProteins of Immunological Interest, 5th edit., US Public Health Service,NIH Bethesda, Md., Publication (1991) No. 91-3242). Structuralcharacteristics used as indices for classification were specifiedaccording to following criteria: Length of CDR1:

The number of amino acids from the 31st amino acid from the N-terminusto one amino acid before the 36th W (tryptophan) that is defined byKabat. Length of CDR2:

The number of amino acids from the 50th amino acid as defined by Kabatto one amino acid before the 66th R (arginine). Cys position number:

When Cys was located in the N-terminal side of the 36th W (tryptophan),the number indicating its position counted from the N-terminus wasdefined as the Cys position number. When Cys was located in theC-terminal side of the 36th W (tryptophan), the position of W36 wasdefined as 36, and the number count up to the Cys position was definedas the Cys position number. TABLE 1 Number of Library from Library fromCysteine germlines primer (SEQ ID primer (SEQ ID residue Length ofLength of according to NO: 10) (mainly NO: 11) (mainly Subfamilyposition CDR2 (aa) CDR1 (aa) existing report* IgG2-derived)IgG3-derived) 1 — 21 5 1 20 2 1 18 5 1 17 4 1 5 1 4 7 6 1 7 1 16 2 1 5 12 2 33 21 5 1 20 5 1 1 19 7 1 18 5 3 3 17 2 1 1 4 2 1 5 5 23 19 6 3 2 71 8 1 10 1 16 5 1 8 6 15 5 1 1 14 5 1 3 30 22 3 1 17 2 3 5 6 16 5 2 9 36 4 45 17 5 6 16 2 1 5 3 7 9 6 1 7 1 8 1 5 32 17 4 1 5 2 2 6 16 5 19 5 16 1 6 50 17 5 2 6 7 1 16 5 6 7 32, 33 17 5 1 6 16 5 6 8 30, 32 17 4 1Total 33 89 59*(EMBO J. 19 (5) 921, 2000)

According to the literature (Nguyen et al., EMBO J. 19(5), 921 (2000)),classification of VHH genes into 7 subfamilies had been attempted basedon:

-   1) position of the cysteine residue, and-   2) length of CDR2 sequence.

Specifically, the reference proposes to classify the genes based on thecysteine residue position (30, 32, 33, or 45) and the length of CDR2sequence (16 or 17 amino acids) as shown in Table 2. TABLE 2 Length ofCysteine residue position CDR2 (aa) none 33 30 45 32 17 1a 2a 5a 16 2b3b 4b 5b

The blanks in the Table mean that the authors of the above-mentionedreference could not find clones corresponding to them. In contrast, asshown in Table 1, diversity greater than that of the subfamilyclassifications shown in Table 2 was found as a result of analysis bythe present inventors. For example, many of the clones had CDR2 lengthsother than 16 or 17 amino acids, and some showed a cysteine residueposition of 50 or a combination of the above-mentioned cysteine residuepositions.

Furthermore, in the above-mentioned literature, CDR1 of 5 amino acids inlength are the only ones reported. However, as apparent from Table 1showing the analysis results by the present inventors, great variety wasalso found in the length of CDR1. Different from CDR3, CDR1 and CDR2 arelocated at positions that are not changed during rearrangement ofantibody genes. Therefore, basically, the lengths of CDR1 and CDR2 areconsidered not to change in-germlines as well as in mRNA.

Considering the above, as shown in Table 1, the present inventorsclassified VHH into 8 subfamilies base on 1) cysteine positions.Furthermore, a new class division was aimed with additional indices of:2) length of CDR2 sequence, and 3) length of CDR1 sequence. The divisioninto each class as in Table 1 shows that there is far greater diversityof germlines than that reported in the literature. Therefore, VHH genesamplified by the primers newly designed by the present inventors can beregarded as more faithfully reproducing the in vivo diversity of camels.

The subfamilies of VHHs newly discovered by the present inventors areuseful as indices of VHH gene diversity of libraries. Specifically, alibrary can be determined to maintain the in vivo diversity, when 33clones arbitrary selected from clones constituting the library, andanalyzed for their VHH structure are classified into at least 8 or moreclasses based on the classification method of the present inventors. Apreferred library of the present invention that maintains the in vivodiversity comprises clones of 6 or more VHH subfamilies as well as 15 ormore classes when sufficient amount of clones are randomly sampled fromclones constituting the library and then investigated.

EXAMPLE 3 Production of VHHs Against GST Using VHH-Type AntibodyLibraries

3-1. Determination of Screening Conditions

Based on the screening method of WO 01/62907, VHHs having bindingaffinity for glutathione S-transferase (GST) were selected.

GST was prepared to a final concentration of 0.1 mg/mL using PBS, 3.8 mLof this solution was placed into each of 1 (first time) or 2 (second andthird time) test tubes (Nunc, MaxiSorp™), and incubated at 4° C. for 18hr to adsorb GST on the inner wall of the test tubes. After adsorption,the solution was discarded, 3.8 mL of PBS containing 2% skim milk wasadded to each tube, and reacted at 25° C. for 1 hr for blocking.

IgG2 and IgG3 libraries were screened independently without mixing. Theamount of phage used is shown under the column of “Input phage (cfu)” inTable 3. 3.8 ml of phage solution suspended in PBS containing 2%skimmilk was added to each test tube, and after reacting at roomtemperature for 2 hr, the test tubes were washed 8 times with PBS-0.05%Tween 20.

Next, 3.5 mL of 0.1 M triethylamine (pH 12.3) was added to each testtube, and reacted in a rotator at room temperature for 20 min fordissociation. Then, neutralization by adding 0.875 mL of 1 M Tris-HClbuffer (pH 6.8) to each test tube allowed collection of phages bound tothe antigen-bound MaxiSorp™ tubes.

3-2. Amplification of Collected Phases

The collected solution was treated as follows: infection of the phageinto E. coli, infection of helper phage, and collection of the phage, topurify and amplify the contained phage.

1) Infection of Phage into E. coli

E. coli (DH12S) was cultured in 50 mL of 2×YT medium, and when theabsorbance at a wavelength of 600 nm reached 0.5, the phage dissociatedin 3-1 was added thereto. This was cultured with shaking at 37° C. for 1hr.

2) Infection of Helper Phage

To 54 mL of the above-mentioned culture of 1), 433 mL of 2×YT medium,12.5 mL of 40% glucose, and 0.5 mL of 100 mg/mL ampicillin were added.After culturing at 37° C. to reach an absorbance of 0.5 at thewavelength of 600 nm, the bacteria were precipitated and collected bycentrifugation at 5,000 rpm for 10 min at 4° C., and then suspended in150 mL of 2×YT medium added with 0.15 mL of 100 mg/mL ampicillin. Tothis suspension, 1/100 amount (1.5 mL) of helperphage M13K07 was addedand cultured with shaking at 37° C. for 1 hr.

The culture was added to 450 mL of medium (2×YT medium containing 100μg/mL of amipicillin and 70 μg/mL of kanamycin) pre-warmed to 37° C. andcultured at 37° C. overnight.

3) Collection of Phage

The culture mentioned above in 2) was centrifuged at 8,000 rpm for 10min at 4° C., and 1/5 volume of 20% polyethylene glycol containing 2.5 Msodium chloride was added to the supernatant. This solution was leftstanding at room temperature for 20 min, centrifuged at 8,000 rpm for 15min at 4° C., and then the precipitate was collected. Sterilized PBS wasadded at 1/10 volume of the culture to dissolve the precipitate. 20%polyethylene glycol containing 2.5 M sodium chloride was added again at1/5 volume of the culture, and this was centrifuged at 10,000 rpm for 20min at 4° C. The supernatant was discarded, and the residue was furtherspun down and then centrifuged at 10,000 rpm for 2 min at 4° C. PBScontaining 0.05% NaN₃ was added at 1/100 volume of the culture todissolve the precipitate, and thus, VHH phage was collected.

3-3. Rescreening using Amplified Phase

Using the amplified phage, similar screening was repeated usingantigen-bound test tubes. Since washing during screening is an importantstep in dissociating non-specifically adsorbed phages to select phageswith high binding ability, the washing conditions were set at 30 timesfor the second screening, and 35 times for the third screening.

3-4. Method for Evaluating Phase Screening

When the value (total number of phages placed into an antigen-adsorbedtest tube)+(total number of phages collected from the antigen-adsorbedtest tube) becomes obviously smaller compared to that for the previousscreening during repeated screening by the method mentioned above, aphage displaying the VHH of interest can be supposed to being moreconcentrated. The number of phage contained in the solution wascalculated as follows:

-   1) Dilution series of phage were produced as follows:

[1] 1×10⁻² dilution: 10 μL of phage solution +990 μL of PBS

[2] 1×10⁻⁴ dilution: 10 μL of the dilution [1]+990 μL of PBS

[3] 1×10⁻⁶ dilution: 10 μL of the dilution [2]+990 μL of PBS

[4] 1×10⁻⁸ dilution: 10 μL of the dilution [3]+990 μL of PBS

[5] 1×10⁻⁹ dilution: 100 μL of the dilution [4]+900 μL of PBS

[6] 1×10⁻¹⁰ dilution: 100 μL of the dilution [5] +900 μL of PBS

990 μL of DH12S was added to 10 μL of [4], [5], and [6] of the dilutionseries and incubated for infection at 37° C. for 1 hr. 100 μL of thesesolutions were spread onto LBGA plates, cultured at 30° C. for 18 to 24hr, and the numbers of colonies were counted. Among the above-mentioneddilution series, usually the plate treated with [4] produced 50 or moreplaques. The number of phages per mL was calculated as shown below basedon the number of plaques on the plate treated with [4]. Number of phagesin the stock solution=(number of colonies/plate)x (1×10⁸)×10³ cfu/mL.

3-5. Results of GST Screening

The number of collected phage was calculated similarly, and the numberof phage displaying VHH against the antigen was determined for perscreening. The results are shown in Table 3. Since the ratio ofcollected phage (input/output) increased in the third screening,specific VHH was expected to be concentrated at this stage. TABLE 3Input phage Output phage Wash (cfu) (cfu) Input/output IgG2 library 1st8 1.00 × 10¹⁴ 1.30 × 10⁹ 7.69 × 10⁴ 2nd 30 3.60 × 10¹⁴ 2.20 × 10⁶ 1.64 ×10⁸ 3rd 35 2.00 × 10¹³ 4.70 × 10⁷ 4.26 × 10⁵ IgG3 library 1st 8 1.00 ×10¹⁴ 1.40 × 10⁹ 7.14 × 10⁴ 2nd 30 3.90 × 10¹⁴ 2.60 × 10⁶ 1.50 × 10⁸ 3rd35 2.00 × 10¹³ 1.20 × 10⁷ 1.67 × 10⁶3-6 Measuring Antigen Binding Activity of VHH Obtained by Screening

3-6-1. Confirming Activity of Obtained Phage VHH by ELISA

The antigen binding activity (affinity) of VHH selected by theabove-mentioned screening was measured by ELISA using 96-well microtiterplate. As the samples, not phage-type VHH but VHH-cp3-type VHH was used.

First, to express VHH-cp3, phage-infected E. coli was cultured in 2×YTcontaining 1% glucose and 100 μg/mL ampicillin at 30° C. for 18 hr, then5 μL of the above-mentioned culture was added to 1.5 mL of 2×YTcontaining 0.1% glucose and 100 μg/mL ampicillin, and was cultured at30° C. for 4 hr. At this time, the concentration of E. coli was measuredas the absorbance at a wavelength_of 600 nm and was approximately 0.5.

To this culture, isopropyl-1-thio-β-D-galactoside (IPTG) was added to aconcentration of 1 mM and cultured at 30° C. for 18 hr. 1.5 mL of theculture was sampled into an Eppendorph tube, and centrifuged at 10,000rpm for 5 min at 4° C. The supernatant was collected and sodium azidewas added at 0.1% to prepare a sample.

Next, GST-bound ELISA plates were prepared. Specifically, GST wasdiluted to a final concentration of 100 μg/mL. 100 μL of the diluted GSTsolution was added to each well of a 96-well microtiter plate (Nunc,MaxiSorp™), and after binding at 4° C. for 18 hr, 200 μL of 5% BSA(blocking solution) was added to each well and blocking was performed at37° C. for 1 hr. After discarding the blocking solution, the plate waswashed once with PBS and used for affinity measurements. 100 μL of thesample was added to each well and reacted at 25° C. for 1 hr. After thereaction, the plate was washed 4 times with PBS, 100 μL of 250-folddiluted peroxidase-labeled anti-cp3 antibody (MBL) was added and reactedat 25° C. for 1 hr. Again, the plate was washed 4 times with PBS, 100 μLof a solution of orthophenylenediamine and hydrogen peroxide was addedand reacted for a while, then 100 μL of 2 N sulfuric acid was added tostop the reaction, and the absorbance at a wavelength of 492 nm wasmeasured. As a result, binding activity was confirmed in 71 clones outof 192 clones. 46 clones of IgG2 (FIG. 7) and 25 clones of IgG3 (FIG. 8)having binding activity were selected.

3-6-2. Sequence Analysis of Obtained Anti-GST VHHs

Seventy-one clones showing antigen binding activity were selected,cultured in LBGA at 30° C. for 18 hr, and phagemids were then purifiedusing KURABO DNA Isolation System PI-50. These phagemids were used toconfirm the nucleotide sequences of their genes. Using fluorescenceprimer T7 (ALOKA), the nucleotide sequences were determined by thedideoxy method using Thermosequence kit (Amersham Pharmacia), and ALOKAL1-COR4200L(S)-2.

Based on their CDR3 sequences, one type of IgG3 and six types of IgG2were confirmed. The CDR3 amino acid sequences of the clones obtained bythe screening are summarized in Table 4. One type of CDR3 in IgG3 isalso found in IgG2, and suggests the existence of amechanism of subclassswitching. TABLE 4 Origin Amino acid sequence Number of clones IgG2 CDR3VFKSWCSDGLGTTLPNY 13 IgG2 CDR3 DFKPWCSDGLGTTLPNY 26 IgG2 CDR3VSGRAYCSGMSIYGDSD 2 IgG2 CDR3 TDESPLRRRFSLLDRRRYD 1 IgG2 CDR3DGGYYSCGVGEE 1 IgG2 CDR3 KSYMCGSTLWRRIDOYND 1 IgG2 CDR3DISAPPGIGGTCAFLGDY 1 IgG3 CDR3 VFKSWCSDGLGTTLPNY 253-7. Purification of Anti-GST VHH (Conversion to Protein A-Fused Type,Expression-Confirming ELISA, Large-Scale Purification)

Three types of anti-GST VHH were converted to protein A-fused proteinsfrom cp3-fused proteins. Following 3 clones were used as anti-GST VHHs.

No. 21: a clone where 2 clones were isolated

No. 29: a clone with the highest ELISA value

NO. 75: a clone comprising the most common CDR3 (38 clones)

The amino acid sequence and nucleotide sequence of each clone are shownin following SEQ ID NOs. Clone No. Nucleotide sequence Amino acidsequence No. 21 SEQ ID NO: 14 SEQ ID NO: 15 No. 29 SEQ ID NO: 16 SEQ IDNO: 17 No. 75 SEQ ID NO: 18 SEQ ID NO: 19

The procedures were as shown below. First, the cp3 region was removed byself ligation using SalI to obtain a transformant. Specifically, each ofthe No. 21, No. 29, and No. 75 clone DNA was SalI-cleaved by followingmethod.

1 μg of DNA (each of the cp3 expression-type VHH expression vectors)/33μL sterilized MILLIQ water

4 μL of 10×High Buffer (Takara, supplied with SalI)

3 μL SalI (Takara)

A reaction solution of the above-mentioned composition was incubated at37° C. for 2 hr. This was electrophoresed on 0.8% agarose gel at 100 mAfor approximately 1 hr, and the band found near 4 kB was cut out using arazor blade. DNA was extracted with QIAEXII (QIAGEN) and subjected toethanol precipitation. Thus obtained DNA was self ligated underfollowing conditions.

Collected DNA dissolved in 62 μL of sterilized MILLIQ water 10 mM DTT 10μL 10 mM ATP 10 μL 10× Ligase buffer 10 μL T4 DNA Ligase (Takara) 350U/μL 8 μL

After incubating the reaction solution at 16° C. for 15 hr, DNA wascollected by ethanol precipitation. This DNA was dissolved in 3 μL of10-fold sterilized MILLIQ water-diluted TE (10 mM Tris, 1 mM EDTA pH8.0). 1.5 μL thereof was used to transform 20 μL of DH12S byelectroporation. The conditions for the transformation were as describedabove in 1-4-5 under the item of “Transformation of heavy chain variableregion antibody libraries (IgG2 and IgG3 libraries) for VHH-typeselection”. The obtained bacterial strain was cultured in 500 mL of 2×TYcontaining 0.1% glucose and 100 μg/mL ampicillin, and expression wasinduced with 1 mM IPTG in the early stage of logarithmic growth (OD 660nm=0.5). Twenty hours after the expression induction, bacteria werecollected to obtain the supernatant as the culture supernatantcomprising protein A-type VHH.

Next, the protein A-type VHH was purified as follows. First, 36 g ofammonium sulfate was added to per 100 mL of the obtained culturesupernatant (60% ammonium sulfate) to precipitate protein by stirring at4° C. for 1 hr. This was then centrifuged at 8,000 rpm for 10 min and-the supernatant was discarded.

One tablet of protease inhibitor (Complete™, Roche) dissolved in 30 mLof PBS was added per 500 mL of the culture supernatant, and theprecipitate was removed by centrifugation at 10,000 rpm for 15 min.0.05% NaN₃ and 1.5 mL of IgG Sepharose™ 6 Fast Flow (Pharmacia) resinwere added to 30 mL of the supernatant and stirred for 1 hr. The resinsolution was charged into a column (10 mL poly-prep, Bio-Rad) by gravityand flushed twice, each time with 10 mL of 0.1% Tween-PBS. Next, 10 mLof PBS each time was flowed twice. Furthermore, 50 mL of PBS was flowedsimilarly. After finally passing 5 mL of 10-fold diluted PBS through,protein A-type VHH was eluted under following conditions.

Three mL of 50 mM citric acid (pH 2.4) was passed through and the eluatewas collected.

Next, 4 mL of 0.1 M glycine (pH 3.0) was passed through and the eluatewas collected.

Finally, 5 mL of 50 mM citric acid (pH 2.4) was passed through and theeluate was collected.

The collected eluate was neutralized with 350 μL of 3 M Tris, and theneutrality of the solution was confirmed with pH paper. Each of theeluate was dialyzed overnight at 4° C. against 3 L of PBS using a 3500MWCO dialysis membrane (PIERCE). After the dialysis, NaN₃ was added to0.05%. The concentration of the obtained purified protein was determinedby measuring the absorbance at 280 nm. The value obtained by dividingthe OD 280 nm by 1.4 gave the protein concentration as μg/mL.Furthermore, the molecular weight (MW) of the collected protein wasconfirmed by SDS-PAGE. The results are as shown below. Proteinconcentration Molecular weight No. 21 445 μg/15 mL MW. 30 KD No. 29 494μg/15 mL MW. 30 KD No. 75 649 μg/15 mL MW. 28 KD

The theoretical molecular weight of a protein A-type VHH is 28 KD.Therefore, the results of SDS-PAGE support the finding that theseproteins have the structure of interest.

Next, using the supernatant of the transformant, the binding activity ofthe protein A-type VHH for GST was confirmed. First, 16 transformantclones were each induced to express the protein in small quantity, andtheir culture supernatant containing protein A-type VHH was dispensedinto GST-coated microwells (MaxiSorp™) as a primary antibody. Then,rabbit anti-mouse Fab antibody (4000-fold dilution) having affinity forProtein A was used as the secondary antibody, and 4000-fold diluted goatanti-rabbit IgG-HRP (MBL) as the tertiary antibody to confirm theexpression of protein A-type VHH and its binding ability.

As a result, in all of the culture supernatants of the transformants,protein A-type VHH having binding activity towards GST was detected.

3-4. Binding Constant Measurement on Purified Anti-GST VHHs

The interaction with GST of VHHs selected from the library of thisinvention was analyzed. Specifically, ka (binding rate constant), kd(dissociation rate constant), and KD (dissociation constant; kd/ka) ofthe interaction with GST of clones No. 75 and No. 29 were determined byaffinity measurement and kinetic analysis. BIAcorel1000 biosensor devicewas used for the analysis.

Carboxymethyldextran (Sensor Chip CM5, Research grade, BIAcore) sensorchip was used. Antigen (GST) was immobilized on the chip by.electrostatic attachment to the CM5 matrix and by covalent bondingbetween the lysyl group of CM5 and activated carboxyl group of theantigen. The carboxyl group was activated by EDC/NHS coupling reaction(Johnson et al.).

At a flow rate of 5 μL/min using HBS-EP (BIAcore), lysyl groups on CM5were activated (2.4 min of contact time) with EDC/NHS (equal amounts ofEDC and NHS of Amine Coupling Kit (BIAcore) were mixed), and then thechip was washed with HBS-EP (BIAcore). Next, 20 μg/mL of GST (Sigma, 0.6mg protein/mL diluted in 10 mM acetic acid (pH 4.0)) was added to thechip. After HBS-EP wash of the chip, 1 Methanol amine (pH 8.5) wasadded, and the remaining activated carboxyl group was inactivated. Afterthe inactivation, the chip was washed with 50 mM NaOH, and all GST thatdid not form covalent bounds were removed. The amount of immobilizedantigen was calculated to be 2252 RU. The binding was evaluated as near300 RU by adding saturating amount of clone No. 75 to the sensor.

All analysis experiments were performed at an HBS-EP flow rate of 35μL/min of HBS-EP (BIAcore) at 25° C., and regeneration in 50 mM NaOH for1 min.

The binding was traced by altering the concentration of clone No. 75(5×10⁻⁸ M to 4.0×10⁻⁷ M). Using (Langmuir) binding (BIAevaluation Ver.3), the curve showed a satisfactory fit. Base line correction was alsoconsidered. When ka (1/Ms) was determined by Global fitting(BIAevaluation Ver. 3), a value of 3.81×10⁴ M⁻¹S⁻¹ was obtained. When kd(1/S) was determined, the value was 9.15×10⁻⁴S⁻¹. Due to the occurrenceof mass transport limitation, this value is used as the lower limit. TheKD value calculated based on kinetic analysis was 24 nM.

When the binding of clone No. 29 was evaluated, the amount ofimmobilized antigen was 1750 RU, and saturating amount of clone No. 29was added to the sensor to give a value of near 460 RU. Similarconditions were used for other parameters of the evaluation. The ka(1/Ms) was 2.54×10⁴M-¹S-¹, kd (1/S) was 2.55×10⁻³S⁻¹, and the KD valuecalculated based on kinetic analysis was 100 nM. The kd of clone No. 75was low, and therefore had a high binding constant. Accordingly, cloneNo. 75 was considered to be a VHH that does not readily dissociate.

EXAMPLE 4 4. Enzyme Activity Inhibition Experiment using PurifiedAnti-GST VHH

4-1. Parameter Setting for Enzyme Activity Measurement

GST is a bisubstrate enzyme that uses both glutathione and CDNB assubstrates. The influence of VHH selected from the VHH libraries of thepresent invention on the enzyme activity was evaluated based on theconditions reported in the literature (Habig WH et al., J. Biol. Chem.Nov. 25, 249(22), 7130 (1974)): 1 mM CDNB, 1 mM glutathione, and 1.5 μMGST. Since the curve was linear up to 3 min from the start of the enzymereaction and decreased somewhat thereafter, the slope was calculatedfrom the value per min up to 3 min to determine the enzyme reactionrate.

Using this method of measurement, the binding constant by Edie plot wasdetermined wherein CDNB was fixed at 1 mM and glutathione concentrationwas varied in the range of 2-0.0625 mM. The binding constant was thesame as previously reported (Measured value of Km =0.48 mM).Furthermore, the appropriate glutathione concentration was determined sothat it falls within a measurable range (approximately up to 1.5 withrespect to a blank sample of water) on a spectrophotometer when CDNB isvaried and the enzyme concentration is kept constant.

4-2. Confirming Enzyme Inhibition by VHH

To measure under enzyme reaction measurement condition of 1 mM CDNB, 1mM glutathione, and 1.42 μM enzyme, VHH was concentrated with Centricon®YM-10 (amicon). As a result, coexistence of 3.8 to 5 μM VHH (clone: No.21, No. 29, or No. 75) became possible. Thus, the enzyme reaction ratewas measured similarly to 4-1. MILLIQ water 220 μL 1 M potassiumphosphate (pH 6.5) 25 μL 100 mM CDNB 2.5 μL 100 mM glutathione 2.5 μL

A mixed solution of 1.0 μg GST/25 μL PBS and each of the VHHs (0 to 5μM) was incubated at room temperature for 1 hr, and the above-mentionedcomposition was added thereto. After addition, the absorbance at 340 nmwas measured at 25° C.

As a result, no enzyme inhibitory activity of No. 21 and No. 29 could beobserved even at the highest VHH concentration. For No. 75, VHHconcentration-dependent enzyme inhibitory activity seemed to exist. Thisinhibition was significant at 1.5 μM or more. The measurement result ofNo. 75 is shown in FIG. 9.

Furthermore, to confirm whether the apparent Km is changing, CDNB waskept constant at 1 mM, and the glutathione concentration was variedbetween 2 to 0.0625 mM. The reaction was performed under followingconditions: MILLIQ water 220 μL 1 M potassium phosphate (pH 6.5) 25 μL100 mM CDNB 2.5 μL 200 mM to 6.25 mM glutathione 2.5 μL

A mixed solution of 1.0 μg GST/25 μL PBS and each of the VHHs (2 μM) wasincubated at room temperature for 1 hr, and the above-mentionedcomposition was added thereto. After addition, the absorbance at 340 nmwas measured at 25° C.

FIG. 10 summarizes a part of the results. In the interest of No. 29,even the result at the highest VHH concentration was nearly the same asthat in the absence of VHH. On the other hand, for No. 75, the slopesignificantly differs with a VHH concentration of 2 μM, where theapparent Vmax was small and the apparent Km was unchanged. This resultshows that the VHH of No. 75 inhibits GST noncompetitive withglutathione.

Next, the Lineweaver-Burk plot obtained with 0.25 mM glutathione andchanging CDNG from 4 to 0.25 mM was confirmed to become linear. Themeasurement was carried out under following conditions: 1 M potassiumphosphate (pH 6.5) 25 μL 100 mM CDNB 10 to 1.25 μL 25 mM glutathione 2.5μL adjusted to 250 μL with MILLIQ water.

A mixed solution of 1.0 μg GST/25 μL PBS, and each of the VHHs (2 μM)was incubated at room temperature for 1 hr, and the above-mentionedcomposition was added thereto. After addition, the absorbance at 340 nmwas measured at 25° C.

A part of the results is summarized in FIG. 11. When No. 75 VHH was usedin this system, the slope changed significantly and the apparent Vmaxbecame small, but the apparent Km did not change. This indicates thatthe VHH inhibited GST noncompetitive with CDNB.

Accordingly, No. 75 VHH acts noncompetitive on both of the substrates.Therefore, theVHHseems to recognize, asepitope, asite of GST that isdifferent from the binding sites of these substrates. Thus, no productsmay be generated from the enzyme substrate complex (ESC), anintermediate, due to its changes in the conformation by the binding ofthe VHH.

EXAMPLE 5 Lactic Acid Dehydrogenase VHH Screening using VHH-TypeAntibody Library

5-1. LDH Screening

VHH having binding affinity for lactic acid dehydrogenase was screened.Bacillus stearothemophillus-derived lactic acid dehydrogenase (L-LACTICDEHYDROGENASE; LDH, SIGMA, 250 units) expressed in E. coli was used asan antigen for the screening.

The active unit of commercially available LDH was described as 123units/mg solid and 586 units/mg protein. The purity of this commerciallyavailable product was confirmed by SDS-PAGE. As a result, the purity wasconsidered to be approximately 80%. (Dimers of two 35-KD monomers andtetramers of 4 of the monomers are formed.) The procedure for screeningwas as follows. LDH concentration was adjusted with PBS to 0.2 mg/mL,and dispensed at 25 μL/well into MaxiSorp™ loose (Nunc-Immuno™ Module).Four wells were used for the first screening, and 1 well each for the2nd to 4th screenings. LDH was adsorbed on the inner walls of the wellsby incubation at 4° C. for 18 hr. After adsorption, the solution wasdiscarded, 150 μL/well of PBS containing 1% BSA was added, and reactedat 37° C. for 1 hr for blocking.

Fifty μL of the library was added at the amount of input phage (cfu)(PBS containing 1% BSA was used as the buffer) shown in Table 5, andafter reacting for 2 hr at 37° C., the wells were washed with PBS by thenumber of times shown in Table 5. Then, phages bound to the antigen werecollected as follows. Specifically, 50 μL/well of 0.1 M HCl-glycine (pH2.2) was added, and reacted at room temperature for 10 min fordissociation. Three μL/well of 2 M Tris was added thereto forneutralization, and the solution was collected.

5-2. Amplification of Collected Phase

The collected solution was treated as follows: infection of the phageinto E. coli, infection of helper phage, and collection of the phages topurify and amplify the comprised phage.

1) Infection of phage into E. coli

E. coli (DH12S) was cultured in 2 mL of 2×YT media to proliferate to anabsorbance of 0.5 at a wavelength of 600 nm. Then, the solution of phagedissociated in 5-2 was added thereto and cultured with shaking at 37° C.for 1 hr.

2) Infection of helper phage

To the culture of 1), 6 mL of Super Broth medium (30 g of Triptone(DIFCO), 20 g of yeast extract (DIFCO), and 10 g of MOPS (NakalaiTesque) were filled up to 1 L using distilled water, pH was adjusted to7.0, and was steam-sterilized at 121° C. for 20 minutes) and 100 mg/mLof ampicillin to a final volume of 1/1000 were added, and cultured withshaking at 160 rpm for 2 hr at 37° C. Subsequently, 1012 CFU (1.0 mL) ofhelper phage M13K07, 92 mL of Super Broth medium, and 100 mg/mL ofampicillin to a final volume of 1/1000 were added thereto, and culturedwith shaking at 160 rpm for 2 hr at 37° C. Kanamycin was then added to aconcentration of 70 μg/mL, and cultured overnight at 37° C.

Collection of phages, re-screening of amplified phages, and method ofevaluating phage screening followed the method described for anti-GSTVHH, except that PBS was used for washing and washing was performed 15times for the 2nd and 3rd screening and 20 times for the 4th screening.

5-3. Result of LDH Screening

The course of screening is shown in Table 5. As apparent from Table 5,the collection rate (output/input) increased in the 4th screening andVHH against LDH was considered to be isolated. TABLE 5 input number ofscreening phage times of output phage cycle number (cfu) washing (cfu)output/input 1   4 × 10¹² 7 1.2 × 10⁷ 1/(3.3 × 10⁵) 2 1.1 × 10¹² 15 2.1× 10⁶ 1/(5.2 × 10⁵) 3 2.3 × 10¹² 15 1.6 × 10⁶ 1/(1.4 × 10⁶) 4 2.3 × 10¹¹20 4.5 × 10⁶ 1/(5.1 × 10⁴)

Similarly to anti-GST VHH, 60 clones and 36 clones were prepared asmonoclones from the 3rd and 4th screenings, respectively. Next, ELISAwas performed similarly as for anti-GST VHH.

LDH dissolved in PBS at 200 μg/mL was used to sensitize MaxiSorp™.Culture supernatant, 10-fold diluted mouse anti-cp3 monoclonal 3G3A8H1,and 1000-fold diluted goat anti-mouse IgG (H+L)-POD were added asprimary, secondary, and tertiary antibodies, respectively, to thisantigen-sensitized plate. As negative control, PBS was added instead ofperforming antigen sensitization.

The results of ELISA are shown in FIG. 12 (the 3rd screening) and FIG.13 (the 4th screening). In the 4th screening, 29 clones were ELISApositive.

Next, the nucleotide sequences of the genes were analyzed by a similarmethod to anti-GST VHH. As a result, 11 types of VHHs, judged from theirCDR3 sequences, were isolated (2 kinds of VH types existed)

5-4. Conversion of Anti-LDH VHH to Protein A-Fused Type

According to the method described in Example 3 (GST), 8 of the cloneswere converted to protein A types by SalI digestion and self-ligation.

Transformants transfected with each of the VHH clones were cultured at800 mL scale, and purified with IgG Sepharose to yield 200 to 800 μg ofpurified VHH for every clone. The nucleotide sequences of the 8 clonesused in the experiment and the amino acids encoded by those nucleotidesequences are shown in the following SEQ ID NOs. Clone No. NucleotideSequence Amino acid sequence No. 407 SEQ ID NO: 20 SEQ ID NO: 21 No. 415SEQ ID NO: 22 SEQ ID NO: 23 No. 418 SEQ ID NO: 24 SEQ ID NO: 25 No. 421SEQ ID NO: 26 SEQ ID NO: 27 No. 426 SEQ ID NO: 28 SEQ ID NO: 29 No. 428SEQ ID NO: 30 SEQ ID NO: 31 No. 430 SEQ ID NO: 32 SEQ ID NO: 33 No. 434SEQ ID NO: 34 SEQ ID NO: 355-5. Inhibitory Activity of Lactic Acid Dehydrogenase VHH

The activity to inhibit the reaction to produce lactic acid from pyruvicacid was detected for clones No. 407, 415, 421, 426, 428, 430, 434, andcontrol. Anti-GST VHH (clone No. 29) obtained in the above Example wasused as the control.

First, VHH concentration was estimated by SDS-PAGE. Then, 8 μM of VHHwas incubated for 1 hr with LDH (1.4 μM), and the residual enzymeactivity was measured. The conditions for activity detection were asfollows:

66 mM sodium phosphate buffer (pH 7.0)

1 mM pyruvic acid

120 μM NADH

280 nM LDH, 1.6 μM VHH (final), 26° C. (room temperature)

The reaction catalyzed by LDH is as follows:LDHpyruvic acid+NADH+H⁺→L-lactic acid+NAD⁺

The enzyme activity was detected by measuring the absorbance at 340 nm.The measurement results are shown in FIG. 14. As a result, the enzymereaction was rather enhanced by clones No. 418, No. 421, and No. 428.No. 430 showed inhibitory activity. No. 407, No. 415, No.426, and No.434showed same reaction rate with anti-GST VHH (clone No. 29) addition thatwas used as a control, and were considered not to affect the LDH enzymeactivity.

Next, considering the possibility of contamination by substrates andpresence of low-molecular-weight reaction-promoting substances, the VHHsolution was filtered through an ultrafiltration membrane with 3000 MWcut-off (Microcon YM-3) and the results were confirmed with thisfiltrate alone. As a result, neither promoting effect nor inhibitoryeffect could be confirmed in the filtrate. Therefore, the action ofregulating enzyme activity confirmed in FIG. 14 was considered to be dueto the activity-regulating effect by a high-molecular-weight protein,i.e., VHH. Under the reaction conditions used herein, VHHs that increasethe reaction rate in the direction of lactic acid production by 10 fold(No. 418 and No. 421), and by 5 fold (No. 428), and a VHH that inhibitsthe reaction rate in the direction of lactic acid production (No. 430)were obtained.

Preliminary experiments with modified pyruvic acid concentration toraise sensitivity were performed to detect enzyme activity usingdecreased amounts of enzyme and VHH. These experiments revealed that theenzyme activity can be measured using the enzyme at 22.4 nM.Accordingly, VHH concentration dependence was investigated for clone No.430. (56 nM of enzyme and the VHH were incubated)

Conditions for measuring the enzyme activity were as below:

66 mM sodium phosphate buffer (pH 7.0)

10 mM pyruvic acid

120 μM NADH

22.4 nM LDH (final), 26° C.

The measurement results are shown in FIG. 15. The inhibitory effect ofVHH could be observed starting from 5 fold (278 nM) of VHH, and with 20fold VHH, the residual activity decreased to 20%.

5-6. Binding Constant Measurement of Lactic Acid Dehydrogenase VHH usingBIAcore

Procedure similar to that with GST was used to immobilize LDH onCM-sensor chip (Sensor Chip CM5, Research grade, BIAcore) using theamino coupling method.

Lysyl groups on CM5 were activated using EDC/NHS (equal amounts of EDCand NHS of Amine Coupling Kit (BIAcore) were mixed) (8-minute contacttime), and the chip was washed with HBS-EP (BIAcore). Twenty μg/mL ofLDH (Sigma, 0.2 μg protein/mL diluted in 10 mM acetic acid (pH5.0)) wasthen added to the chip. After washing the chip with HBS-EP, 1 M ethanolamine (pH 8.5) was added to inactivate the remaining activated carboxylgroups. Following the inactivation, the chip was washed with HBS-EP toremove all LDHs that had not been covalently bound. The amount ofimmobilized antigens was calculated 5653 RU. When binding was evaluatedby adding saturating amounts of clone No. 430 to the sensor, the valuewas near 800 RU. The value was near 500 RU for clone No. 428, and near1400 RU for clone No. 421.

All experiments were performed in HBS-EP at 25° C., and regeneration wasperformed under optimum conditions. Washing was performed for 1 minusing 50 mM citric acid (pH2.5).

The binding was monitored by changing the LDH concentration (5×10⁻⁸ M to4.0×10⁻⁷ M). The curve showed a satisfactory fit when (Langmuir) bindingor 1:1 binding with mass transfer (BIAevaluation Ver.3) was used.Base-line correction was also taken into consideration. Values of ka(1/Ms), kd (1/S), and KD calculated based on kinetic analysis usingglobal fitting (BIAevaluation Ver. 3) were as shown in Table 6.

In enzyme inhibition experiments, IC50 of No. 430 was approximately 400nM, close to the measured value of approximately 250 nM for KD. Thisresult was suggested to prove the occurrence of inhibition due to thebinding. TABLE 6 KD × 10⁻⁷ KA × 10⁶ Clone No. ka (M⁻¹S⁻¹) kd (S⁻¹) (M)(1/M) (Langmuir) binding model 421 2.39 × 10⁴ 4.09 × 10⁻³ 1.72 5.83 4281.44 × 10⁴ 5.13 × 10⁻³ 3.56 2.81 430 9.82 × 10³ 2.58 × 10⁻³ 2.63 3.801:1 binding with mass transfer model 421 2.39 × 10⁴ 4.09 × 10⁻³ 1.715.83 428 1.48 × 10⁴ 5.16 × 10⁻³ 3.50 2.86 430 1.15 × 10⁴ 2.85 × 10⁻³2.48 4.04

EXAMPLE 6 6. Construction of IgM Heavy Chain Variable Region Library

6-1. Cloning of IgM Constant Region, Cμ (constant μ)

Similar to Example 1, cDNA was prepared from mRNA of 22 camels usingrandom primers.

The following primer was constructed based on the sequence of theC-terminal portion of IgM constant region, Cμ (constant μ), which isconserved among humans and mice. PCR was performed using the N-terminalprimer of the heavy chain variable region and camel cDNA to clone camelCμ. AACGTAGGCGCGCCGGACTTGTCCACGGTCCTCTC/ SEQ ID NO: 38

The underlined indicates the AscI cleavage site.

PCR fragments were cleaved with SfiI and AscI restriction enzymes,cloned into pFCA-10 vector cleaved with SfiI and AscI, and then used fortransformation by the aforementioned method. DNA was prepared asdescribed above from the transformants, and the nucleotide sequence ofthe N-terminal region of Cμ was determined. The nucleotide sequence andamino acid sequence of the N terminus of camel Cμ are shown below.E   S   S   S   A   P   T   L   Y   P   L   / SEQ ID NO: 40 GAG AGC TCATCT GCC CCG ACA CTC TTC CCC CTC/ SEQ ID NO: 396-2. Construction of IgM Heavy Chain Variable Region

An oligonucleotide comprising the following nucleotide sequence wasdesigned as a primer that can selectively yield the IgM heavy chainvariable region from the newly cloned camel Cμ sequence.ACATTAATCTGGCGCGCCGAGAGTGTCGGGGCAGATG SEQ ID NO: 41 AGCTCTC/

By a method similar to that previously described in the section of IgGVHH library construction, cDNA was obtained using random primers with 20μg of the obtained mRNA from 22 camels as a template.

Using 1/40 amount of the obtained cDNA as a template, each of the 6types of primers (SEQ ID NOs: 1 to 6) of the N-terminal region of the Vdomain was combined with the above-mentioned primer, and PCR wasperformed using 6 types of primer sets.

PCR conditions were 95° C. for 3 min, followed by 19 cycles of 94° C.for 1 min, 72° C. for 2 min, and 72° C. for 1 min. All PCR productsobtained from each combination of primers were combined, electrophoresedon 0.8% agarose gel, and a band at approximately 0.5 kbp was cut outusing a razor blade. QIAEX II (QIAGEN) was used for DNA extraction. Theamount of collected DNA estimated using DAPI was 178 μg.

6-3. Cloning of IgM Heavy Chain Variable Region Into Vector

According to the method described in Example 1, the mixed collected PCRfragments were cleaved with SfiI restriction enzyme, ligated toSfiI-cleaved pFCA-10 vector, and DNA fragments were collected byperforming phenol treatment and ethanol precipitation. The fragmentswere further cleaved with AscI restriction enzyme, collected by phenoltreatment and ethanol precipitation, and ligation was performed. Theamount of the collected DNA measured by DAPI staining was 66 μg.

6-4. Transformation

According to the method described in Example 1, the DNA obtained abovewas used to transform 7.5 mL of DHS12S. (The process of transforming 20μL of ElectroMAX^(m) DH12S with 0.17 μg of the DNA by electroporationwas repeated.) The overall number of transformed bacteria estimated bysampling a part of this material was 6.6×10¹⁰.

6-5. Construction of IgM heavy chain variable region library phage

Overnight culture of a part of the transformed E. coli resulted in2.5×10¹¹ DH12S/87.5 mL. Two liter of sterilized 2×TY media, glucose at afinal concentration of 1%, and ampicillin at a final concentration of100 μg/mL were added thereto, and cultured at 37° C. until OD 600 nmreached 0.8. The culture was centrifuged at 8,000 rpm for 10 min at 4°C., and bacterial cells in the precipitate were dissolved in 2 L ofsterilized 2×TY media containing ampicillin at a final concentration of100 μg/mL. Twenty milliliter of helper phage K07 was then added theretoand cultured at 37° C. for 1 hr.

Four liter of sterilized 2×TY media, ampicillin at a final concentrationof 100 μg/mL, and kanamycin at a final concentration of 50 μg/mL wereadded thereto and cultured overnight. According to the method describedin Example 1, phages were collected from the overnight culture, yielding50 mL of 2.4×1014 cfu/mL phage library.

EXAMPLE 7 7. Screening of Antibodies Against β-Gal using IgM Heavy ChainVariable Region Library

7-1. Conditions for Screening Antibodies Against β-Gal

The method for screening was performed similarly to that of GSTaccording to WO 01/62907 and Example 3. However, β-gal concentration wasadjusted to 0.1 mg/mL using PBS, 3.8 mL of the β-gal solution was addedto each of the 2 test tubes (Nunc, MaxiSorp™) (1st screening) or 1 testtube (2nd and 3rd screenings), and incubated at 4° C. for 18 hr toadsorb β-gal on the inner walls of the tubes. After adsorption, thesolution was discarded, 3.8 mL of PBS containing 2% skim milk was addedto each tube, and reacted at 25° C. for 1 hr for blocking.

The IgM heavy chain variable region library was screened using theamount of input phage (cfu) shown in following Table, and adding 3.8 mLof PBS containing 2% skim milk to each test tube. (Table 7)

7-2. Results of Screening for Antibodies Against β-Gal

The collection rate (output/input) increased in the 3rd screening, andantibodies against β-gal were considered to be concentrated. TABLE 7Screening Number of cycle Input phage times of Output phage number (cfu)washing (cfu) Output/Input 1 1.0 × 10¹³ 8 4.0 × 10⁹ 1/(3.0 × 10⁴) 2 3.5× 10¹² 31 4.7 × 10⁵ 1/(7.4 × 10⁶) 3 1.4 × 10¹² 31 7.8 × 10⁹ 1/(180)7-3. Anti- β-Gal Antibody Monoclone ELISA

Monoclones were prepared from phage clones obtained from the thirdscreening by a method similar to that for GST antibodies of Example 3.When ELISA was performed by a method similar to that for GST antibodiesof Example 3, 43 clones out of 48 clones were ELISA positive. (FIG. 18)

Industrial Applicability

The present invention provides VHH libraries that maintain the in vivodiversity of the VHH region. Furthermore, VHHs constituting thelibraries of the present invention comprise many normal nucleotidesequences, and they have a high expression rate. Thus, it can be statedthat the VHH libraries of this invention are composed of active VHHs.Therefore, using a VHH library of the present invention, a VHH havingbinding affinity for any arbitrary antigen can be freely obtained.

In general, camelids that have not been immunologically sensitized hadbeen considered to provide only VHHs having binding affinity forantigens with strong immunogenicity. However, the present inventorsrealized a VHH library that freely provides VHHs having a wide varietyof functions by enlarging the repertoire size of the library. Thepresent invention greatly contributes to the industrial use of VHHs byproviding libraries that require no immunological sensitization andhaving richer diversity compared to known libraries.

On the other hand, conventional methods for constructing libraries didtake no account of limitations or bias of in vivo VHH repertoire size inan individual. As a result, the diversity of VHH libraries constructedby conventional methods did not reflect the in vivo diversity of the VHHregion. This is also obvious from the fact that many of the VHH classesclassified in Table 1 have structural characteristics newly found in thelibraries of the present invention. Methods to increase the diversity oflibraries by artificially introducing mutations are well known in theart. However, the methods of artificially introducing mutationsaccompany inefficiency of producing far too many inactive antibodiesalong with the production of active antibodies. As a result, VHHlibraries constructed based on conventional methods did not fulfill therequirement of a VHH library, i.e., composed of active VHHs with richdiversity.

A VHH has excellent characteristics in solubility and stability comparedto that of a generally used VH constituting IgG. Furthermore, a bindingactivity difficult to obtain for IgG composed of VHs can be expectedfrom VHHs. However, according to the findings of the present inventors,the in vivo repertoire size of VHHs in an individual is restricted.Therefore, as long as libraries are constructed based on conventionallibrary production methods, the repertoire size of a VHH library cannotexceed the in vivo repertoire size of an individual. Thus, althoughvarious utilities are expected from VHHs, the possibility to obtain aVHH having the desired function is extremely low when known VHHlibraries are utilized.

The present invention enables to freely obtain industrially useful VHHsby providing VHH libraries having a diversity exceeding the in vivorepertoire size of an individual. In other words, VHHs having thedesired-function were made accessible for the first time due to thelibraries of the present invention.

This is also apparent from the result of the Examples wherein VHHshaving the function of regulating enzyme activity are easily selected.Furthermore, in the Examples, a plurality of VHHs having various effectson a plurality of enzymes was selected. The great diversity of thelibraries of the present invention as well as the activity of VHHdifferent from that of VH enables to readily select such VHHs havingvarious functions.

In addition, a library consisting of IgM-derived VHs provided by thepresent invention is useful as a library that additionally supplementsthe above-mentioned VHH libraries having diversity. Therefore, the useof an IgM-derived VH library of the present invention enables selectionof antibody variable regions that cannot be selected from VHHs or thosehaving functions that are difficult to select due to small populationsize.

1. A library of camelid-derived VHHs, which maintains the in vivodiversity of variable regions in a camelidae.
 2. The library of claim 1,wherein 33 arbitrary clones selected from clones constituting thelibrary comprise genes belonging to at least 8 or more classes.
 3. Thelibrary of claim 2, wherein a sufficient amount of clones randomlyselected from clones constituting the library comprise genes of at least6 VHH subfamilies, and at the same times genes belonging to 15 or moreclasses.
 4. The library of claim 1 comprising at least 10⁵ or more VHHgene clones.
 5. The library of claim 1 consisting of VHH gene clonesderived from immunoglobulin genes of IgG2 and/or IgG3.
 6. The library ofclaim 5, wherein the VHH gene rate in the library is 60% or more.
 7. Thelibrary of claim 1, which is an rgdp library.
 8. A method of obtaining agene encoding a VHH that has an affinity for a substance of interest,which comprises the steps of: (1) contacting the library of claim 7 withthe substance of interest, and (2) selecting a clone encoding a VHH thatbinds to the substance of interest.
 9. The method of claim 8, whereinthe substance of interest is an enzyme molecule or a fragment thereof.10. A method of obtaining a VHH that has a function to regulate anenzyme activity, which comprises the steps of: (1) obtaining a VHH thatbinds to an enzyme by the method of claim 9, (2) contacting the VHHobtained in step (1) with the enzyme, and (3) selecting the VHH that hasa function to modify the enzyme activity of the enzyme compared to thatin the absence of the VHH.
 11. A gene encoding a VHH selected by themethod of claim 8 or
 10. 12. A method of producing an immunoglobulincomprising a camelid-derived VHH as a variable region, or a fragmentthereof, which comprises the steps of: (1) obtaining a gene encoding aVHH that has a binding activity for a substance of interest by themethod of claim 8, (2) preparing a VHH expression vector byincorporating the obtained VHH-encoding gene into a vector expressiblein a host cell, and (3) introducing the VHH expression vector into thehost cell to collect proteins comprising the VHH from the culture.
 13. Amethod of constructing a VHH library, which comprises the steps of: (1)obtaining VHH genes from a plurality of animals belonging to Camelidae,and (2) preparing a library by mixing the VHH genes obtained in step(1).
 14. The method of claim 13 comprising the step of amplifying theVHH genes obtained in step (1).
 15. The method of claim 14, wherein theamplification is performed by PCR.
 16. The method of claim 15 comprisingthe step of collecting amplification products of the PCR during theexponential phase.
 17. The method of claim 15, wherein the animals ofCamelidae are dromedaries, and the PCR is performed using primer setsconsisting of a 5′ primer selected from any one of the oligonucleotideshaving the nucleotide sequences of SEQ ID NOs: 1 to 6 and a 3′ primerconsisting of an oligonucleotide having the nucleotide sequence of SEQID NO: 10, and which comprises the step of mixing amplification productsfrom each of the primer sets.
 18. The method of claim 15, wherein theanimals of Camelidae are dromedaries, and the PCR is performed usingprimer sets consisting of a 5′ primer selected from any one of theoligonucleotides having the nucleotide sequences of SEQ ID NOs: 1 to6and a 3′ primer consisting of an oligonucleotide having the nucleotidesequence of SEQ ID NO: 11, and which comprises the step of mixingamplification products from each of the primer sets.
 19. The method ofclaim 17 or 18 comprising the step of digesting the amplificationproducts with restriction enzymes SfiI and AscI and ligating thedigested products into a vector having features (i) and (ii) as follows:(i) comprising a SfiI site and an AscI site; and (ii) upontransformation of the vector into an appropriate host, expressing aprotein encoded by an exogenous gene inserted into the site of (i) as afusion protein with a protein constituting a phage.
 20. A VHH library,which can be constructed by the method of claim 17 or
 18. 21. A primerset for camel VHH gene amplification consisting of a 5′ primer selectedfrom oligonucleotides having the nucleotide sequences of SEQ ID NOs: 1to 6, and a 3′ primer selected from oligonucleotides having thenucleotide sequences of SEQ ID NOs:10 and 11, respectively.
 22. A methodof constructing a VH library, which comprises the steps of: (1)obtaining VH genes from a plurality of animals of Camelidae, and (2)preparing a library by mixing the VH genes obtained in step (1).
 23. Themethod of claim 22 comprising the step of amplifying the VH genesobtained in step (1).
 24. The method of claim 23, wherein theamplification is performed by PCR;
 25. The method of claim 24 comprisingthe step of collecting the amplification products of the PCR during theexponential phase.
 26. The method of claim 24, wherein the animals ofCamelidae are dromedaries, and the PCR is performed using primer setsconsisting of a 5′ primer selected from any one of the oligonucleotideshaving the nucleotide sequences of SEQ ID NOs: 1 to 6 and a 3′ primerconsisting of an oligonucleotide having the nucleotide sequence of SEQID NO: 41, and which comprises the step of mixing the amplificationproducts from each of the primer sets.
 27. The method of claim 26comprising the step of digesting the amplification products withrestriction enzymes SfiI and AscI and ligating the digested product intoa vector having features (i) and (ii) as follows: (i) comprising a SfiIsite and an AscI site, and (ii) upon transformation of the vector intoan appropriate host, expressing a protein encoded by an exogenous geneinserted into the site of (i) as a fusion protein with a proteinconstituting a phage.
 28. A VH library derived from camelid IgM.
 29. AVH library obtainable by the method of claim
 22. 30. The library ofclaim 28 or 29, which is an rgdp library.
 31. A method of obtaining agene encoding a VH that has an affinity for a substance of interest,which comprises the steps of: (1) contacting the library of claim 30with the substance of interest, and (2) selecting a clone comprising aVH that binds to the substance of interest;
 32. The method of claim 31,wherein the substance of interest is an enzyme molecule or a fragmentthereof.
 33. A method of obtaining a VH comprising a function toregulate an enzyme activity, which comprises the steps of: (1) obtaininga VH that binds to an enzyme by the method of claim 31, (2) contactingthe VH obtained in step (1) with the enzyme, and (3) selecting the VHthat has a function to modify the enzyme activity of the enzyme comparedto that in the absence of the VH.
 34. A gene encoding a VH selected bythe method of claim
 31. 35. A method of producing an immunoglobulincomprising a dromedary-derived VH as a variable region, or a fragmentthereof, which comprises the steps of: (1) obtaining a gene encoding aVH having a binding activity for a substance of interest by the methodof [31], (2) preparing a VH expression vector by incorporating theobtained VH-encoding gene into a vector expressible in a host cell, and(3) introducing the VH expression vector into the host cell, andcollecting proteins comprising the VH from the culture.
 36. A primer setfor dromedary VH gene amplification that consists of a 5′ primerselected from any one of the oligonucleotides having the nucleotidesequences of SEQ ID NOs:1 to 6, and a 3′ primer consisting of anoligonucleotide having the nucleotide sequence of SEQ ID NO: 41.